Anaplasma phagocytophilum (Aph) antigens and antibodies specific for Anaplasma

ABSTRACT

The invention provides methods and compositions for the detection and treatment of  Anaplasma phagocytophilum  and  Anaplasma platys  infection.

PRIORITY

This application is a divisional application of U.S. Ser. No.12/745,047, filed on Sep. 8, 2010, now U.S. Pat. No. 8,158,370, which isa national stage entry of International Application No.PCT/US2008/084391, filed on Nov. 21, 2008, which claims the benefit ofthe filing date of U.S. Provisional Application Ser. No. 60/990,420,filed Nov. 27, 2007. The entire contents of the foregoing applicationsare incorporated by reference herein in their entirety.

SEQUENCE LISTING

This document incorporates by reference herein an electronic sequencelisting text file, which is filed in electronic format via EFS-Web. Thetext file is named “07-060-WO-US.seqlist.txt,” is 1,770 bytes, and wascreated on Apr. 12, 2012.

BACKGROUND OF THE INVENTION

Anaplasmosis occurs in numerous mammal species such as humans, horses,dogs, cats deer, and ruminants and is caused by infection ofgranulocytic cells with the tick-borne agent Anaplasma phagocytophilum(“Aph”) (formerly known as Ehrlichia equi). Frequently reported clinicalsigns in granulocytic ehrlichiosis in humans are leukopenia andthrombocytopenia. Common clinical signs in dogs are fever,thrombocytopenia, swelling of the lymph nodes, and anorexia.

Anaplasma platys (“Apl”) (formerly known as Ehrlichia platys) is anotheragent that is likely transmitted through ticks such as Rhipicephalussanguineus or other arthropod. A. platys may be co-transmitted by a tickwith Ehrlichia canis. A. platys can cause infectious canine cyclicthrombocytopenia (ICCT), but infected dogs are usually asymptomatic. A.platys infection is difficult to detect in vivo because the numbers ofthe bacteria in the blood are usually low. Serologic tests for Apl canbe inaccurate because of cross-reactivity with other Anaplasma sp.

Tickborne infectious disease caused by A. phagocytophilum in human anddogs is a serious problem, whereas A. platys infection is presentlyconsidered to be of minor importance. Current serodiagnostic tools forA. phagocytophilum can not differentiate the two infections. Methods ofdetecting Aph and Apl and methods of differentiating between the twoinfections are needed in the art.

The onset of clinical symptoms occurs during the acute phase ofanaplasmosis—typically within 7 to 14 days post infection—and canprecede the advent of measurable levels of antibodies against some Aphantigens. Thus, there is a need for a rapid, sensitive and reliableimmunological test for Aph infection in mammals exhibiting clinicalsymptoms of acute anaplasmosis.

SUMMARY OF THE INVENTION

One embodiment of the invention provides a purified polypeptide that isat least 95% identical to SEQ ID NO:1, 2, 6, 8, 9, 10, 11, or 12,wherein the purified polypeptide consists of less than about 300, 150,100, or 50 contiguous naturally occurring Anaplasma phagocytophilumamino acids. The polypeptide can specifically binds an antibody specificfor Anaplasma phagocytophilum. The purified polypeptide can be linked toan indicator reagent, an amino acid spacer, an amino acid linker, asignal sequence, a stop transfer sequence, a transmembrane domain, aprotein purification ligand, a heterologous polypeptide, one or moreadditional polypeptides comprising SEQ ID NO:1, 2, 6, 8, 9, 10, 11, 12or a combination thereof. The invention provides isolatedpolynucleotides that encode the purified polypeptides of the invention.

Another embodiment of the invention provides a method of detectingantibodies that specifically bind an Anaplasma phagocytophilumpolypeptide in a test sample. The method comprises contacting one ormore purified polypeptides at least 95% identical to SEQ ID NO:1, 9 or11, with the test sample, under conditions that allowpolypeptide/antibody complexes to form. The purified polypeptides canconsist of less than about 150 contiguous naturally occurring Anaplasmaphagocytophilum amino acids. The polypeptide/antibody complexes aredetected. The detection of the polypeptide/antibody complexes is anindication that antibodies specific for Anaplasma phagocytophilum arepresent in the test sample, and the lack of detection of thepolypeptide/antibody complexes is an indication that antibodies specificfor Anaplasma phagocytophilum are not present in the test sample. Thecomplexes can be contacted with an indicator reagent prior to thedetection step. The amount of antibodies in the test sample can bedetermined. The one or more purified polypeptides can be attached to asubstrate. The one or more purified polypeptides can be linked to anindicator reagent, an amino acid spacer, an amino acid linker, a signalsequence, a stop transfer sequence, a transmembrane domain, a proteinpurification ligand, a heterologous protein, one or more additionalpolypeptides comprising SEQ ID NO:1, 9, or 11, or a combination thereof.

Even another embodiment of the invention provides a method of detectingan Anaplasma phagocytophilum infection in a subject. The methodcomprises obtaining a biological sample from the subject; contacting oneor more purified polypeptides that are at least 95% identical to SEQ IDNO:1, 9 or 11, wherein the purified polypeptides consist of less thanabout 150 contiguous naturally occurring Anaplasma phagocytophilum aminoacids, with the biological sample under conditions that allowpolypeptide/antibody complexes to form; and detecting thepolypeptide/antibody complexes. The detection of thepolypeptide/antibody complexes is an indication that the subject has anAnaplasma phagocytophilum infection and the lack of detection of thepolypeptide/antibody complexes is an indication that the subject doesnot have an Anaplasma phagocytophilum infection.

Still another embodiment of the invention provides an antibody thatspecifically binds to a polypeptide consisting of SEQ ID NO:1, 2, 6, 8,9, 10, 11, or 12. The antibody can be a monoclonal antibody, polyclonalantibody, a Fab fragment, a Fab′ fragment, Fab′-SH fragment, F(ab′)₂fragment, Fv fragment, or a single chain antibody.

Yet another embodiment of the invention provides a method of detectingan Anaplasma phagocytophilum polypeptide in a sample. The methodcomprises contacting one or more antibodies that specifically bind to apolypeptide consisting of SEQ ID NO:1, 9, or 11 with the sample underconditions that allow polypeptide/antibody complexes to form, anddetecting the polypeptide/antibody complexes. The detection of thepolypeptide/antibody complexes is an indication that an Anaplasmaphagocytophilum polypeptide is present in the sample and the lack ofdetection of the polypeptide/antibody complexes is an indication that anAnaplasma phagocytophilum polypeptide is not present in the sample. Theone or more antibodies can be monoclonal antibodies, polyclonalantibodies, Fab fragments, Fab′ fragments, Fab′-SH fragments, F(ab′)₂fragments, Fv fragments, or single chain antibodies.

Even another embodiment of the invention provides a method of detectingantibodies that specifically bind an Anaplasma platys polypeptide, anAnaplasma phagocytophilum polypeptide or both an Anaplasma platyspolypeptide and an Anaplasma phagocytophilum polypeptide. The methodcomprises contacting one or more purified polypeptides that are at least95% identical to SEQ ID NO:2, 6, 8, 10, or 12 with a test sample, underconditions that allow polypeptide/antibody complexes to form, anddetecting the polypeptide/antibody complexes. The detection of thepolypeptide/antibody complexes is an indication that antibodies specificfor Anaplasma platys and/or Anaplasma phagocytophilum are present in thetest sample, and the lack of detection of the polypeptide/antibodycomplexes is an indication that antibodies specific for Anaplasma platysand/or Anaplasma phagocytophilum are not present in the test sample. Theamount of antibodies in the test sample can be determined. The one ormore purified polypeptides can be attached to a substrate. The one ormore purified polypeptides can be linked to an indicator reagent, anamino acid spacer, an amino acid linker, a signal sequence, a stoptransfer sequence, a transmembrane domain, a protein purificationligand, a heterologous protein, one or more additional polypeptidescomprising SEQ ID NO:1, 2, 6, 8, 9, 10, 11, 12 or a combination thereof.

Another embodiment of the invention provides a method of detecting anAnaplasma phagocytophilum and/or Anaplasma platys infection in asubject. The method comprises obtaining a biological sample from thesubject; contacting one or more purified polypeptides comprising SEQ IDNO:2, 6, 8, 10, or 12 with the biological sample under conditions thatallow polypeptide/antibody complexes to form; and detecting thepolypeptide/antibody complexes. The detection of thepolypeptide/antibody complexes is an indication that the subject has anAnaplasma phagocytophilum and/or Anaplasma platys infection and the lackof detection of polypeptide/antibody complexes is an indication that thesubject does not have an Anaplasma phagocytophilum and/or Anaplasmaplatys infection.

Even another embodiment of the invention provides a method of detectingan Anaplasma phagocytophilum polypeptide, an Anaplasma platyspolypeptide, or an Anaplasma phagocytophilum polypeptide and anAnaplasma platys polypeptide, in a sample. The method comprisescontacting one or more antibodies that specifically bind to apolypeptide consisting of SEQ ID NO:2, 6, 8, 10, or 12 with the sampleunder conditions that allow polypeptide/antibody complexes to form, anddetecting the polypeptide/antibody complexes. The detection of thepolypeptide/antibody complexes is an indication that an Anaplasmaphagocytophilum polypeptide, an Anaplasma platys polypeptide, or anAnaplasma phagocytophilum polypeptide and an Anaplasma platyspolypeptide is present in the sample and the lack of detection of thepolypeptide/antibody complexes is an indication that an Anaplasmaphagocytophilum and an Anaplasma platys polypeptide is not present inthe sample.

Still another embodiment of the invention provides a method of detectingAnaplasma platys polypeptides and Anaplasma phagocytophilum polypeptidesin a sample. The method comprises:

-   -   (a) contacting one or more antibodies that specifically bind to        a polypeptide consisting of SEQ ID NO:2, 6, 8, 10, or 12 with        the sample under conditions that allow polypeptide/antibody        complexes to form and detecting the polypeptide/antibody        complexes; and    -   (b) contacting one or more antibodies that specifically bind to        a polypeptide consisting of SEQ ID NO:1, 9, or 11 with the        sample under conditions that allow polypeptide/antibody        complexes to form and detecting the polypeptide/antibody        complexes;    -   (c) wherein if the polypeptide/antibody complexes are detected        in step (a) and in step (b) then the sample contains Anaplasma        phagocytophilum polypeptides and may also contain Anaplasma        platys polypeptides; wherein if the polypeptide/antibody        complexes are detected in step (a) and are not detected in        step (b) then the sample contains Anaplasma platys polypeptides        and does not contain Anaplasma phagocytophilum polypeptides;        wherein if the polypeptide complexes are not detected in        step (a) and are not detected in step (b) then the sample does        not contain Anaplasma platys polypeptides and does not contain        Anaplasma phagocytophilum polypeptides.

Another embodiment of the invention provides a method of detectingantibodies that specifically bind an Anaplasma platys polypeptide, anAnaplasma phagocytophilum polypeptide, or both an Anaplasma platyspolypeptide and an Anaplasma phagocytophilum polypeptide. The methodcomprises:

-   -   (a) contacting one or more purified polypeptides that are at        least 95% identical to SEQ ID NO:2, 6, 8, 10, or 12 with a test        sample, under conditions that allow polypeptide/antibody        complexes to form and detecting the polypeptide/antibody        complexes;    -   (b) contacting one or more purified polypeptides that are at        least 95% identical to SEQ ID NO:1, 9, or 11, wherein the        purified polypeptide consists of less than about 150 contiguous        naturally occurring Anaplasma phagocytophilum amino acids, with        a test sample, under conditions that allow polypeptide/antibody        complexes to form and detecting the polypeptide/antibody        complexes. If the polypeptide/antibody complexes are detected in        step (a) and in step (b) then the sample contains antibodies the        specifically bind Anaplasma phagocytophilum polypeptides and        Anaplasma platys polypeptides and antibodies that specifically        bind only Anaplasma platys polypeptides; wherein if the        polypeptide/antibody complexes are detected in step (a) and are        not detected in step (b) then the sample contains antibodies        that specifically bind Anaplasma platys polypeptides and        Anaplasma phagocytophilum polypeptides and does not contain        antibodies that specifically bind only Anaplasma phagocytophilum        polypeptides; wherein if the polypeptide complexes are not        detected in step (a) and are not detected in step (b) then the        sample does not contain antibodies specific for Anaplasma platys        polypeptides and does not contain antibodies specific for        Anaplasma phagocytophilum polypeptides.

Even another embodiment of the invention provides a compositioncomprising a purified polypeptide of the invention and apharmaceutically-acceptable or veterinarily acceptable carrier. Thecomposition can further comprise an adjuvant.

Still another embodiment of the invention provides an immunogencomprising a polypeptide having at least 95% identity to SEQ ID NO:1, 2,6, 8, 9, 10, 11, or 12, and one or more additional regions or moietiescovalently joined to the polypeptide at the carboxyl terminus or aminoterminus, wherein each region or moiety has at least one of thefollowing properties: enhances the immune response, facilitatespurification, or facilitates polypeptide stability.

Yet another embodiment of the invention provides a method of treating orameliorating Anaplasma platys infection, Anaplasma phagocytophiluminfection, or both in a mammalian subject. The method comprisesadministering to the mammalian subject a therapeutically effectiveamount of a composition comprising a purified polypeptide of theinvention and a pharmaceutically-acceptable or veterinarily acceptablecarrier. The composition can further comprise an adjuvant.

Another embodiment of the invention provides a method of inducing animmune response in a mammal comprising administering to the mammal animmunologically effective amount of a composition comprising a purifiedpolypeptide of the invention and a pharmaceutically-acceptable orveterinarily acceptable carrier. The composition can further comprise anadjuvant.

Still another embodiment of the invention provides a first purifiedpolypeptide that specifically binds an antibody, wherein the antibodyspecifically binds a second purified polypeptide, wherein the secondpurified polypeptide consists of SEQ ID NO:1, 2, 6, 8, 9, 10, 11, or 12.The first purified polypeptide can consist of at least 10, but less than300 contiguous amino acids of SEQ ID NO: 1, 2, 6, 8, 9, 10, 11 or 12.The first purified polypeptide can be at least 95% identical to SEQ IDNO: 1, 2, 6, 8, 9, 10, 11 or 12. The first purified polypeptide can belinked to an indicator reagent, an amino acid spacer, an amino acidlinker, a signal sequence, a stop transfer sequence, a transmembranedomain, a protein purification ligand, a heterologous polypeptide, oneor more additional polypeptides comprising SEQ ID NO:1, 2, 6, 8, 9, 10,11, 12, or a combination thereof.

The invention therefore provides methods and compositions for thedetection and treatment of Apl and/or Aph infection.

DETAILED DESCRIPTION OF THE INVENTION

Anaplasma Phagocytophilum Polypeptides

The singular forms “a,” “an”, and “the” used herein include pluralreferents unless the context clearly dictates otherwise.

A polypeptide is a polymer of two or more amino acids covalently linkedby amide bonds. A polypeptide can be post-translationally modified. Apurified polypeptide is a polypeptide preparation that is substantiallyfree of cellular material, other types of polypeptides, chemicalprecursors, chemicals used in synthesis of the polypeptide, etc, orcombinations thereof. A polypeptide preparation that is substantiallyfree of cellular material, culture medium, chemical precursors,chemicals used in synthesis of the polypeptide, etc. has less than about30%, 20%, 10%, 5%, 1% or more of other polypeptides, culture medium,chemical precursors, and/or other chemicals used in synthesis.Therefore, a purified polypeptide is about 70%, 80%, 90%, 95%, 99% ormore pure. A purified polypeptide does not include unpurified orsemi-purified cell extracts or mixtures of polypeptides that are lessthan 70% pure.

The term “polypeptides” can refer to one or more of one type ofpolypeptide (a set of polypeptides). “Polypeptides” can also refer tomixtures of two or more different types of polypeptides (a mixture ofpolypeptides). The terms “polypeptides” or “polypeptide” can each alsomean “one or more polypeptides.”

One embodiment of the invention provides an Aph polypeptide as shown inSEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQID NO:11.

(clone14) SEQ ID NO: 1 LCATVHHIYQ GNYEDRNNDK GSSRGGGTTY YPMTMSASASEESLSSIISE GXLSKTSLPS YSAATATGTG NXTGEVXSHSHSSGKSSSKP ESRPESNLQN VVAETMSQQQ RSVSWherein the X at position 52 is D or G; the X at position 72 is E or A;and the X at position 77 is absent or is F.

(clone13) SEQ ID NO: 2 HKGVDSDRKH DAEKTEEKKH GLGSLCKSLA INLVSLMGTALVXTPIILLA VVLLVLVPVY LLCATVHHIY QGNYEDRNNDKGSSRGGGTT YYPMTMSASA SEESLSSIIS EGXLSKTSLPSYSAATATGT GNXTGEVXSH SHSSGKSSSK PESRPESNLQ NVVAETMSQQ QRSVSWherein the X at position 43 is A or T; wherein the X at position 113 isD or G; wherein the X at position 133 is A or E; and wherein the X atposition 138 is F or absent.

(clone13ext) SEQ ID NO: 8 CLGGKSPART TEERVAGDLD HKGVDSDRKH DAEKTEEKKHGLGSLCKSLA INLVSLMGTA LVXTPIILLA VVLLVLVPVYLLCATVHHIY QGNYEDRNND KGSSRGGGTT YYPMTMSASASEESLSSIIS EGXLSKTSLP SYSAATATGT GNXTGEVXSHSHSSGKSSSK PESRPESNLQ NVVAETMSQQ QRSVSWherein the X at position 63 is A or T; wherein the X at position 133 isD or G; wherein the X at position 153 is A or E; and wherein the X atposition 158 is F or absent.

(p37-1) SEQ ID NO: 9 CATVHHIYQG NYEDRNNDKG SSRGGGTTYY PMTMSASASE ESL(p37-2) SEQ ID NO: 10 CLGGKSPART TEERVAGDLD HKGVDSDRKH DAEKTEEKKH G(p37-3) SEQ ID NO: 11 XXTGEVXSHS HSSGKSSSKP ESRPESNLQN VVAETWherein the X at position 1 is C or absent; wherein the X at position 2is A or E; and wherein the X at position 7 is F or absent.

One embodiment of the invention provides a purified polypeptidecomprising SEQ ID NOs:1, 9 or 11 wherein the polypeptide consists ofless than about 150, 140, 130, 120, 110, 90, 80, 70, 60, 50, 40, 35, 30,25 or 20 (or any range between 150 and 20) contiguous naturallyoccurring Anaplasma phagocytophilum amino acids. Another embodiment ofthe invention provides a purified polypeptide comprising SEQ ID NOs:1, 9or 11 wherein the polypeptide consists of more than about 20, 25, 30,35, 40, 50, 60, 70, 80, 90, 110, 120, 130, 140, or 150 (or any rangebetween 20 and 150) contiguous naturally occurring Anaplasmaphagocytophilum amino acids. Naturally occurring Aph amino acids are anypolypeptides naturally produced by an Aph organism. That is, a purifiedpolypeptide contains a polypeptide shown in SEQ ID NO:1, 9, or 11, butcontains less than about 150, 140, 130, 120, 110, 90, 80, 70, 60, 50,40, 35, 30, 25 or 20 contiguous naturally occurring Anaplasmaphagocytophilum amino acids. That is, the purified polypeptide issmaller than the full length polypeptide. The purified polypeptide cancomprise additional heterologous amino acids (that is, amino acids thatare not from Aph) and even additional Aph amino acids as long as they donot naturally occur contiguously with SEQ ID NOs:1, 2, 8, 9, 10, or 11.Another embodiment provides a purified polypeptide comprising SEQ IDNO:2, 6, 8, 10, or 12 wherein the polypeptide consists of less thanabout 350, 300, 275, 250, 225, 200, 195, 175, 150, 125, 100, 75, 50, 45,40, 35, 30, or 25 (or any range between 350 and 25) contiguous naturallyoccurring Anaplasma phagocytophilum amino acids. That is, the purifiedpolypeptide is smaller than the full length polypeptide (SEQ ID NO:6).Another embodiment provides a purified polypeptide comprising SEQ IDNO:2, 6, 8, 10, or 12 wherein the polypeptide consists of more thanabout 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 195, 200, 225,250, 275, 300 or 350 (or any range between 25 and 350) contiguousnaturally occurring Anaplasma phagocytophilum amino acids.

In one embodiment of the invention, a purified polypeptide can compriseSEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11, wherein the polypeptideconsists of more than about 200, 175, 150, 160, 150, 140, 130, 120, 114,110, 100, 90, 80, 70, 60, 50, 43, 40, 35, 30, or 25 (or any rangebetween 200 and 25) contiguous naturally occurring Anaplasmaphagocytophilum amino acids. Another embodiment provides a purifiedpolypeptide comprising SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11,wherein the polypeptide consists of less than about 200, 175, 150, 160,150, 140, 130, 120, 114, 110, 100, 90, 80, 70, 60, 50, 43, 40, 35, 30,or 25 (or any range between 200 and 25) contiguous naturally occurringAnaplasma phagocytophilum amino acids. The fact that these polypeptidesare smaller than the full length Aph polypeptide APH_(—)0915 (GenBankAccession Number ABD43857) (SEQ ID NO:6) is important because smallerpolypeptides can have greater specificity and/or sensitivity than fulllength polypeptides in Apl and/or Aph assays. Additionally, thesesmaller polypeptides can be less expensive to manufacture, and may beobtained at greater purity, than the full length polypeptide.

(APH_0915 hypothetical protein {Anaplasma phagocytophilum HZ})SEQ ID NO: 6 MSFTMSKLSLDPTQGSHTAENIACSIFDMVLGVKSTAKLLAGTWAGTSSTIWKTVTGAASSTKEASSKSYGTLRSSLGSSASRRMLGTCATAALCLTAPLLGAAAAGAAITCALITICMALLFLVLYTVLHIASQMLRCASLLLSMVCNILHSTFTATKSCLGGKSPARTTEERVAGDLDHKGVDSDRKHDAEKTEEKKHGLGSLCKSLAINLVSLMGTALVTTPIILLAVVLLVLVPVYLLCATVHHIYQGNYEDRNNDKGSSRGGGTTYYPMTMSASASEESLSSIISEGGLSKTSLPSYSAATATGTGNATGEVFSHSHSSGKSSSKPESRPESNLQNVV AETMSQQQRSVS

Another embodiment of the invention provides a polypeptide shown in SEQID NO:12:

SEQ ID NO: 12MSFTMSKLSL DPTQGSHTAE NIACSIFDMV LGVKSTAKLL AGTWAGTSST IWKTVTGAAS 60STKEASSKSY GTLRSSLGSS ASRRMLGTCA TAALCLTAPL LGAAAAGAAI TCALITICMA 120LLFLVLYTVL HIASQMLRCA SLLLSMVCNI LHSTFTATKS CLGGKSPART TEERVAGDLD 180HKGVDSDRKH DAEKTEEKKH GLGSLCKSLA INLVSLMGTA LVXTPIILLA VVLLVLVPVY 240LLCATVHHIY QGNYEDRNND KGSSRGGGTT YYPMTMSASA SEESLSSIIS EGXLSKTSLP 300SYSAATATGT GNXTGEVXSH SHSSGKSSSK PESRPESNLQ NVVAETMSQQ QRSVSwherein the X at position 223 is A or T, the X at position 293 is D orG, the X at position 313 is E or A and the X at position 318 is F orabsent.

One embodiment of the invention provides a purified polypeptide that isless than about 360, 350, 300, 250, 200, 175, 150, 125, 100, 90, 80, 70,60, 50, 40, 35, 30, 25, 20, or 10 contiguous naturally Aph amino acidsand greater than about 10, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,125, 150, 175, 200, 250, 300, 350, or 360 contiguous amino acids of SEQID NOs:1-2, 6, and 8-10 (or any range between 360 and 10 amino acids).

One embodiment of the invention provides a purified polypeptidecomprising at least about 10, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 360 or morecontiguous amino acids of SEQ ID NOs:1-2, 6, and 8-12. Therefore, apolypeptide of the invention can be, for example, about 19 to about 40;about 19 to about 50; about 19 to about 100; or about 19 to about 150amino acids in length.

Variant polypeptides are at least about 79% or 80%, or about 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%identical to the polypeptide sequences shown in SEQ ID NOs:1-2, 6, and8-12 and are also polypeptides of the invention. For example, a variantpolypeptide of SEQ ID NO:11 can be about at least 97% (about 1 aminoacid change), 94% (about 2 amino acid changes), 91% (about 3 amino acidchanges), 89% (about 4 amino acid changes), 86% (about 5 amino acidchanges), identical to SEQ ID NO:11.

One embodiment of the invention provides a purified polypeptide that isless than 150 contiguous amino acids and greater than about 10, 20, 30,35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 or 140 contiguous aminoacids of SEQ ID NO:6 (or SEQ ID NO:6 having an A at position 223, a D atposition 293, an E at position 313, and no amino acid at position 318).For example, a purified polypeptide of the invention is less than about150 contiguous amino acids of SEQ ID NO:6 (or SEQ ID NO:6 having an A atposition 223, a D at position 293, an E at position 313, and no aminoacid at position 318) and greater than about 30 contiguous amino acidsof SEQ ID NO:6 (or SEQ ID NO:6 having an A at position 223, a D atposition 293, an E at position 313, and no amino acid at position 318).Purified polypeptides of the invention can comprise, e.g., amino acids150-355, 161-355, 181-355, 242-355 and fragments thereof of aboutgreater than 10, 20, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130or 140 contiguous amino acids of SEQ ID NO:6 (or SEQ ID NO:6 having an Aat position 223, a D at position 293, an E at position 313, and no aminoacid at position 318).

One embodiment of the invention provides a purified polypeptidecomprising at least about 10, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 190 or more contiguous aminoacids of SEQ ID NOs:1, 2, 6, 8, 9, 10, 11, or 12.

Another embodiment of the invention provides a purified polypeptidecomprising SEQ ID NO:1, 9, or 11, wherein the polypeptide consists ofless than about 150 contiguous naturally occurring Anaplasmaphagocytophilum amino acids or a purified polypeptide comprising SEQ IDNO: 2, 6, 8, 10, or 12, wherein the polypeptide consists of less thanabout 300 contiguous naturally occurring Anaplasma phagocytophilum aminoacids.

Variant polypeptides at least about 80, or about 90, 91, 92, 93, 94, 95,96, 98, or 99% identical to the polypeptide sequences shown in SEQ IDNOs:1, 2, 6, 8, 9, 10, 11, or 12 are also polypeptides of the invention.Variant polypeptides have one or more conservative amino acid variationsor other minor modifications and retain biological activity, i.e., arebiologically functional equivalents. A biologically active equivalenthas substantially equivalent function when compared to the correspondingwild-type polypeptide. In one embodiment of the invention a polypeptidehas 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50 conservative amino acidsubstitutions.

Percent sequence identity has an art recognized meaning and there are anumber of methods to measure identity between two polypeptide orpolynucleotide sequences. See, e.g., Lesk, Ed., Computational MolecularBiology, Oxford University Press, New York, (1988); Smith, Ed.,Biocomputing: Informatics And Genome Projects, Academic Press, New York,(1993); Griffin & Griffin, Eds., Computer Analysis Of Sequence Data,Part I, Humana Press, New Jersey, (1994); von Heinje, Sequence AnalysisIn Molecular Biology, Academic Press, (1987); and Gribskov & Devereux,Eds., Sequence Analysis Primer, M Stockton Press, New York, (1991).Methods for aligning polynucleotides or polypeptides are codified incomputer programs, including the GCG program package (Devereux et al.,Nuc. Acids Res. 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul et al.,J. Molec. Biol. 215:403 (1990)), and Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711) whichuses the local homology algorithm of Smith and Waterman (Adv. App.Math., 2:482-489 (1981)). For example, the computer program ALIGN whichemploys the FASTA algorithm can be used, with an affine gap search witha gap open penalty of −12 and a gap extension penalty of −2.

When using any of the sequence alignment programs to determine whether aparticular sequence is, for instance, about 95% identical to a referencesequence, the parameters are set such that the percentage of identity iscalculated over the full length of the reference polynucleotide and thatgaps in identity of up to 5% of the total number of nucleotides in thereference polynucleotide are allowed.

Variant polypeptides can generally be identified by modifying one of thepolypeptide sequences of the invention, and evaluating the properties ofthe modified polypeptide to determine if it is a biological equivalent.A variant is a biological equivalent if it reacts substantially the sameas a polypeptide of the invention in an assay such as animmunohistochemical assay, an enzyme-linked immunosorbent Assay (ELISA),a radioimmunoassay (RIA), immunoenzyme assay or a western blot assay,e.g. has 90-110% of the activity of the original polypeptide. In oneembodiment, the assay is a competition assay wherein the biologicallyequivalent polypeptide is capable of reducing binding of the polypeptideof the invention to a corresponding reactive antigen or antibody byabout 80, 95, 99, or 100%. An antibody that specifically binds acorresponding wild-type polypeptide also specifically binds the variantpolypeptide.

A conservative substitution is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. In general, the following groups of amino acids representconservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr;(2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg,his; and (5) phe, tyr, trp, his.

A polypeptide of the invention can further comprise a signal (or leader)sequence that co-translationally or post-translationally directstransfer of the protein. The polypeptide can also comprise a linker orother sequence for ease of synthesis, purification or identification ofthe polypeptide (e.g., poly-His), or to enhance binding of thepolypeptide to a solid support. For example, a polypeptide can beconjugated to an immunoglobulin Fc region or bovine serum albumin.

A polypeptide can be covalently or non-covalently linked to an aminoacid sequence to which the polypeptide is not normally associated within nature, i.e., a heterologous amino acid sequence. A heterologousamino acid sequence can be from a non-Anaplasma phagocytophilumorganism, a synthetic sequence, or an Anaplasma phagocytophilum sequencenot usually located at the carboxy or amino terminus of a polypeptide ofthe invention. Additionally, a polypeptide can be covalently ornon-covalently linked to compounds or molecules other than amino acids,such as indicator reagents. A polypeptide can be covalently ornon-covalently linked to an amino acid spacer, an amino acid linker, asignal sequence, a stop transfer sequence, a transmembrane domain, aprotein purification ligand, or a combination thereof. A polypeptide canalso be linked to a moiety (i.e., a functional group that can be apolypeptide or other compound) that enhances an immune response (e.g.,cytokines such as IL-2), a moiety that facilitates purification (e.g.,affinity tags such as a six-histidine tag, trpE, glutathione, maltosebinding protein), or a moiety that facilitates polypeptide stability(e.g., polyethylene glycol; amino terminus protecting groups such asacetyl, propyl, succinyl, benzyl, benzyloxycarbonyl ort-butyloxycarbonyl; carboxyl terminus protecting groups such as amide,methylamide, and ethylamide). In one embodiment of the invention aprotein purification ligand can be one or more C amino acid residues at,for example, the amino terminus or carboxy terminus or both termini of apolypeptide of the invention. An amino acid spacer is a sequence ofamino acids that are not associated with a polypeptide of the inventionin nature. An amino acid spacer can comprise about 1, 5, 10, 20, 100, or1,000 amino acids.

If desired, a polypeptide of the invention can be part of a fusionprotein, which can also contain other amino acid sequences, such asamino acid linkers, amino acid spacers, signal sequences, TMR stoptransfer sequences, transmembrane domains, as well as ligands useful inprotein purification, such as glutathione-S-transferase, histidine tag,and Staphylococcal protein A, or combinations thereof. More than onepolypeptide of the invention can be present in a fusion protein. Apolypeptide of the invention can be operably linked to non-Anaplasmaphagocytophilum proteins or Anaplasma phagocytophilum proteins to formfusion proteins. A fusion protein of the invention can comprise one ormore of Anaplasma phagocytophilum polypeptides of the invention,fragments thereof, or combinations thereof. A fusion protein does notoccur in nature. The term “operably linked” means that the polypeptideof the invention and the other polypeptides are fused in-frame to eachother either to the N-terminus or C-terminus of the polypeptide of theinvention.

Polypeptides of the invention can be in a multimeric form. That is, apolypeptide can comprise one or more copies of an Anaplasmaphagocytophilum polypeptide of the invention or a combination thereof. Amultimeric polypeptide can be a multiple antigen peptide (MAP). Seee.g., Tam, J. Immunol. Methods, 196:17-32 (1996).

Polypeptides of the invention can comprise an antigen that is recognizedby an antibody specific for Anaplasma phagocytophilum. The antigen cancomprise one or more epitopes (i.e., antigenic determinants). An epitopecan be a linear epitope, sequential epitope or a conformational epitope.Epitopes within a polypeptide of the invention can be identified byseveral methods. See, e.g., U.S. Pat. No. 4,554,101; Jameson & Wolf,CABIOS 4:181-186 (1988). For example, a polypeptide of the invention canbe isolated and screened. A series of short peptides, which togetherspan an entire polypeptide sequence, can be prepared by proteolyticcleavage. By starting with, for example, 100-mer polypeptide fragments(or smaller fragments), each fragment can be tested for the presence ofepitopes recognized in an ELISA. For example, in an ELISA assay anAnaplasma phagocytophilum polypeptide, such as a 100-mer polypeptidefragment, is attached to a solid support, such as the wells of a plasticmulti-well plate. A population of antibodies are labeled, added to thesolid support and allowed to bind to the unlabeled antigen, underconditions where non-specific absorption is blocked, and any unboundantibody and other proteins are washed away. Antibody binding isdetected by, for example, a reaction that converts a colorless substrateinto a colored reaction product. Progressively smaller and overlappingfragments can then be tested from an identified 100-mer to map theepitope of interest.

A polypeptide of the invention can be produced recombinantly. Apolynucleotide encoding a polypeptide of the invention can be introducedinto a recombinant expression vector, which can be expressed in asuitable expression host cell system using techniques well known in theart. A variety of bacterial, yeast, plant, mammalian, and insectexpression systems are available in the art and any such expressionsystem can be used. Optionally, a polynucleotide encoding a polypeptidecan be translated in a cell-free translation system. A polypeptide canalso be chemically synthesized or obtained from Anaplasmaphagocytophilum cells.

An immunogenic polypeptide of the invention can comprise an amino acidsequence shown in SEQ ID NOs:1, 2, 6, 8, 9, 10, 11, 12 or fragmentsthereof. An immunogenic polypeptide can elicit antibodies or otherimmune responses (e.g., T-cell responses of the immune system) thatrecognize epitopes of a polypeptide having SEQ ID NOs:1, 2, 6, 8, 9, 10,11, or 12. An immunogenic polypeptide of the invention can also be afragment of a polypeptide that has an amino acid sequence shown in SEQID NOs:1, 2, 6, 8, 9, 10, 11, or 12. An immunogenic polypeptide fragmentof the invention can be about 6, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80,90, 100, 150, 175, 200, 250, 300, 350 or more amino acids in length (orany range between 6 and 350). An immunogenic polypeptide of theinvention can be about 350, 300, 250, 200, 175, 150, 125, 100, 90, 80,70, 60, 50, 40, 30, 25, 20, 15, 10, 6, or less amino acids in length (orany range between 350 and 6).

Anaplasma Phagocytophilum Polynucleotides

Polynucleotides of the invention contain less than an entire microbialgenome and can be single- or double-stranded nucleic acids. Apolynucleotide can be RNA, DNA, cDNA, genomic DNA, chemicallysynthesized RNA or DNA or combinations thereof. The polynucleotides canbe purified free of other components, such as proteins, lipids and otherpolynucleotides. For example, the polynucleotide can be 50%, 75%, 90%,95%, 96%, 97%, 98%, 99%, or 100% purified. A nucleic acid moleculeexisting among hundreds to millions of other nucleic acid moleculeswithin, for example, cDNA or genomic libraries, or gel slices containinga genomic DNA restriction digest are not to be considered an isolatedpolynucleotide. The polynucleotides of the invention encode thepolypeptides of the invention described above. In one embodiment of theinvention the polynucleotides encode a polypeptide shown in SEQ IDNOs:1, 2, 6, 8, 9, 10, 11, 12 or fragments thereof. Examples ofpolynucleotides of the invention include: SEQ ID NOs 3, 4, 5, and 7.

(clone14) SEQ ID NO: 3TTATGCGCTA CAGTGCACCA CATCTATCAA GGAAATTACG AAGATCGCAA CAACGACAAA GGTAGCTCCCGTGGCGGCGG TACTACATAT TATCCAATGA CAATGTCTGC AAGTGCTTCT GAAGAGTCCC TTAGCAGCATAATATCTGAA GGAGNTTTGA GTAAGACATC GCTACCAAGT TACTCCGCAG CCACTGCTAC AGGTACTGGAAATGNAACTG GTGAGGTTTN NNCACATTCT CATTCATCTG GTAAAAGTAG CAGCAAACCA GAATCTCGCCCTGAGAGCAA TCTACAGAAT GTGGTAGCAG AAACCATGTC GCAGCAACAA AGGAGCGTCT CCWherein the N at position 155 is A or G; wherein the N at position 215is A or C; and wherein the three N's at positions 230-232 are all T's orare all absent.

(clone 13) SEQ ID NO: 4CACAAAGGGG TGGATTCAGA TCGGAAGCAT GATGCAGAGA AAACAGAAGA GAAAAAACAT GGTTTGGGTAGCCTCTGCAA ATCACTCGCG ATAAATCTGG TCTCCTTAAT GGGAACAGCG CTAGTTNCCA CACCCATAATACTACTTGCA GTAGTTCTAT TAGTGTTGGT GCCAGTATAT CTGTTATGCG CTACAGTGCA CCACATCTATCAAGGAAATT ACGAAGATCG CAACAACGAC AAAGGTAGCT CCCGTGGCGG CGGTACTACA TATTATCCAATGACAATGTC TGCAAGTGCT TCTGAAGAGT CCCTTAGCAG CATAATATCT GAAGGAGNTT TGAGTAAGACATCGCTACCA AGTTACTCCG CAGCCACTGC TACAGGTACT GGAAATGNAA CTGGTGAGGT TTNNNCACATTCTCATTCAT CTGGTAAAAG TAGCAGCAAA CCAGAATCTC GCCCTGAGAG CAATCTACAG AATGTGGTAGCAGAAACCAT GTCGCAGCAA CAAAGGAGCG TCTCCWherein the N at position 127 is G or A; wherein the N at position 338is A or G; wherein the N at position 398 is A or C; wherein the threeN's at positions 413-415 are all T's or are absent.

(APH_0915 hypothetical gene {Anaplasma phagocytophilum HZ}) SEQ ID NO: 5TTGAGTTTTACAATGTCGAAGTTATCGCTTGACCCTACTCAGGGCTCACATACAGCAGAGAATATTGCTTGTTCTATCTTTGATATGGTACTTGGTGTAAAGTCCACTGCAAAACTGTTAGCAGGTACGTGGGCTGGTACAAGCAGCACTATTTGGAAGACAGTAACAGGAGCAGCTTCCTCAACTAAAGAAGCGTCATCAAAGTCGTATGGAACCCTACGTAGTTCCTTGGGCTCTTCCGCTTCTAGAAGGATGCTAGGAACTTGCGCTACCGCCGCTCTCTGCTTAACTGCACCTTTGCTTGGCGCAGCCGCTGCCGGAGCGGCAATAACATGTGCCTTGATAACCATTTGCATGGCTTTGCTGTTCCTCGTTTTGTACACCGTACTCCACATTGCCTCTCAGATGTTGCGTTGTGCATCGCTACTGTTGAGCATGGTATGCAATATCCTGCACAGCACATTCACCGCAACTAAGTCTTGCCTCGGAGGTAAGTCACCTGCGCGAACAACTGAAGAGCGGGTAGCTGGGGATTTAGATCACAAAGGGGTGGATTCAGATCGGAAGCATGATGCAGAGAAAACAGAAGAGAAAAAACATGGTTTGGGTAGCCTCTGCAAATCACTCGCGATAAATCTGGTCTCCTTAATGGGAACAGCGCTAGTTACCACACCCATAATACTACTTGCAGTAGTTCTATTAGTGTTGGTGCCAGTATATCTGTTATGCGCTACAGTGCACCACATCTATCAAGGAAATTACGAAGATCGCAACAACGACAAAGGTAGCTCCCGTGGCGGCGGTACTACATATTATCCAATGACAATGTCTGCAAGTGCTTCTGAAGAGTCCCTTAGCAGCATAATATCTGAAGGAGGTTTGAGTAAGACATCGCTACCAAGTTACTCCGCAGCCACTGCTACAGGTACTGGAAATGCAACTGGTGAGGTTTTTTCACATTCTCATTCATCTGGTAAAAGTAGCAGCAAACCAGAATCTCGCCCTGAGAGCAATCTACAGAATGTGGTAGCAGAAACCATGTCGCAGCAACAAAGGAGCGTCTCC (clone 13ext) SEQ ID NO: 7TGCCTCGGAG GTAAGTCACC TGCGCGAACA ACTGAAGAGC GGGTAGCTGG GGATTTAGAT CACAAAGGGGTGGATTCAGA TCGGAAGCAT GATGCAGAGA AAACAGAAGA GAAAAAACAT GGTTTGGGTA GCCTCTGCAAATCACTCGCG ATAAATCTGG TCTCCTTAAT GGGAACAGCG CTAGTTNCCA CACCCATAAT ACTACTTGCAGTAGTTCTAT TAGTGTTGGT GCCAGTATAT CTGTTATGCG CTACAGTGCA CCACATCTAT CAAGGAAATTACGAAGATCG CAACAACGAC AAAGGTAGCT CCCGTGGCGG CGGTACTACA TATTATCCAA TGACAATGTCTGCAAGTGCT TCTGAAGAGT CCCTTAGCAG CATAATATCT GAAGGAGNTT TGAGTAAGAC ATCGCTACCAAGTTACTCCG CAGCCACTGC TACAGGTACT GGAAATGNAA CTGGTGAGGT TTNNNCACAT TCTCATTCATCTGGTAAAAG TAGCAGCAAA CCAGAATCTC GCCCTGAGAG CAATCTACAG AATGTGGTAG CAGAAACCATGTCGCAGCAA CAAAGGAGCG TCTCCWherein the N at position 187 is G or A; wherein in the N at position398 is A or G; wherein the N at position 458 is A or C; wherein thethree N's at positions 473-475 are all T's or are all absent.

Polynucleotides of the invention can consist of less than about 900,825, 750, 675, 600, 500, 450, 420, 390, 360, 330, 300, 225, 150, 120, or90 (or any range between 900 and 90) contiguous, naturally occurring Aphpolynucleotides. Polynucleotides of the invention can consist of greaterthan about 90, 120, 150, 225, 300, 330, 360, 390, 420, 450, 500, 600,675, 750, 825, 900 or more (or any range between 90 and 900) contiguous,naturally occurring Aph polynucleotides. The purified polynucleotidescan comprise additional heterologous nucleotides (that is, nucleotidesthat are not from Aph) and even additional Aph amino acids as long asthey do not naturally occur contiguously with SEQ ID NOs:3, 4, 5, or 7.Polynucleotides of the invention can comprise other nucleotidesequences, such as sequences coding for linkers, signal sequences, TMRstop transfer sequences, transmembrane domains, or ligands useful inprotein purification such as glutathione-S-transferase, histidine tag,and Staphylococcal protein A. One embodiment of the invention provides apurified polynucleotide comprising at least about 6, 10, 15, 20, 25, 30,40, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, or morecontiguous nucleotides of SEQ ID NOs:3, 4, 5, 7 or other polynucleotidesthat encode polypeptides shown in SEQ ID NOs:1, 2, 6, 8, 9, 10, 11, 12.

Polynucleotides of the invention can be isolated. An isolatedpolynucleotide is a naturally-occurring polynucleotide that is notimmediately contiguous with one or both of the 5′ and 3′ flankinggenomic sequences that it is naturally associated with. An isolatedpolynucleotide can be, for example, a recombinant DNA molecule of anylength, provided that the nucleic acid sequences naturally foundimmediately flanking the recombinant DNA molecule in anaturally-occurring genome is removed or absent. Isolatedpolynucleotides also include non-naturally occurring nucleic acidmolecules. A nucleic acid molecule existing among hundreds to millionsof other nucleic acid molecules within, for example, cDNA or genomiclibraries, or gel slices containing a genomic DNA restriction digest arenot to be considered an isolated polynucleotide.

Polynucleotides of the invention can also comprise fragments that encodeimmunogenic polypeptides. Polynucleotides of the invention can encodefull-length polypeptides, polypeptide fragments, and variant or fusionpolypeptides.

Degenerate nucleotide sequences encoding polypeptides of the invention,as well as homologous nucleotide sequences that are at least about 80,or about 90, 96, 98, or 99% identical to the polynucleotide sequences ofthe invention and the complements thereof are also polynucleotides ofthe invention. Percent sequence identity can be calculated as describedin the “Polypeptides” section. Degenerate nucleotide sequences arepolynucleotides that encode a polypeptide of the invention or fragmentsthereof, but differ in nucleic acid sequence from the wild-typepolynucleotide sequence, due to the degeneracy of the genetic code.Complementary DNA (cDNA) molecules, species homologs, and variants ofAnaplasma phagocytophilum polynucleotides that encode biologicallyfunctional Anaplasma phagocytophilum polypeptides also are Anaplasmaphagocytophilum polynucleotides.

Polynucleotides of the invention can be isolated from nucleic acidsequences present in, for example, a biological sample, such as blood,serum, saliva, or tissue from an infected individual. Polynucleotidescan also be synthesized in the laboratory, for example, using anautomatic synthesizer. An amplification method such as PCR can be usedto amplify polynucleotides from either genomic DNA or cDNA encoding thepolypeptides.

Polynucleotides of the invention can comprise coding sequences fornaturally occurring polypeptides or can encode altered sequences that donot occur in nature. If desired, polynucleotides can be cloned into anexpression vector comprising expression control elements, including forexample, origins of replication, promoters, enhancers, or otherregulatory elements that drive expression of the polynucleotides of theinvention in host cells. An expression vector can be, for example, aplasmid, such as pBR322, pUC, or ColE1, or an adenovirus vector, such asan adenovirus Type 2 vector or Type 5 vector. Optionally, other vectorscan be used, including but not limited to Sindbis virus, simian virus40, alphavirus vectors, poxvirus vectors, and cytomegalovirus andretroviral vectors, such as murine sarcoma virus, mouse mammary tumorvirus, Moloney murine leukemia virus, and Rous sarcoma virus.Minichromosomes such as MC and MC1, bacteriophages, phagemids, yeastartificial chromosomes, bacterial artificial chromosomes, virusparticles, virus-like particles, cosmids (plasmids into which phagelambda cos sites have been inserted) and replicons (genetic elementsthat are capable of replication under their own control in a cell) canalso be used.

Methods for preparing polynucleotides operably linked to an expressioncontrol sequence and expressing them in a host cell are well-known inthe art. See, e.g., U.S. Pat. No. 4,366,246. A polynucleotide of theinvention is operably linked when it is positioned adjacent to or closeto one or more expression control elements, which direct transcriptionand/or translation of the polynucleotide.

Polynucleotides of the invention can be used, for example, as probes orprimers, for example, PCR primers, to detect the presence of Anaplasmaphagocytophilum and/or Anaplasma platys polynucleotides in a testsample, such as a biological sample. Probes are molecules capable ofinteracting with a target nucleic acid, typically in a sequence specificmanner, for example, through hybridization. Primers are a subset ofprobes that can support an enzymatic manipulation and that can hybridizewith a target nucleic acid such that the enzymatic manipulation occurs.A primer can be made from any combination of nucleotides or nucleotidederivatives or analogs available in the art that do not interfere withthe enzymatic manipulation.

A probe or primer can be about 6, 10, 15, 20, 25, 30, 40, 50, 75, 100,200, 300, 400, 500, 600, 700, 800, 900, 1,000, or more contiguousnucleotides of SEQ ID NOs:3, 4, 5, 7 or other polynucleotides thatencode polypeptides shown in SEQ ID NOs:1, 2, 6, and 8-12.

The hybridization of nucleic acids is well understood in the art anddiscussed herein. Typically a probe can be made from any combination ofnucleotides or nucleotide derivatives or analogs available in the art.The ability of such probes and primers to specifically hybridize toAnaplasma phagocytophilum polynucleotide sequences will enable them tobe of use in detecting the presence of complementary sequences in agiven test sample. Polynucleotide probes and primers of the inventioncan hybridize to complementary sequences in a test sample such as abiological sample, including saliva, sputum, blood, plasma, serum,urine, feces, cerebrospinal fluid, amniotic fluid, wound exudate, ortissue. Polynucleotides from the sample can be, for example, subjectedto gel electrophoresis or other size separation techniques or can beimmobilized without size separation. The polynucleotide probes orprimers can be labeled. Suitable labels, and methods for labeling probesand primers, are known in the art, and include, for example, radioactivelabels incorporated by nick translation or by kinase, biotin labels,fluorescent labels, chemiluminescent labels, bioluminescent labels,metal chelator labels and enzyme labels. The polynucleotides from thesample are contacted with the probes or primers under hybridizationconditions of suitable stringencies.

Depending on the application, varying conditions of hybridization can beused to achieve varying degrees of selectivity of the probe or primertowards the target sequence. For applications requiring highselectivity, relatively stringent conditions can be used, such as lowsalt and/or high temperature conditions, such as provided by a saltconcentration of from about 0.02 M to about 0.15 M salt at temperaturesof from about 50° C. to about 70° C. For applications requiring lessselectivity, less stringent hybridization conditions can be used. Forexample, salt conditions from about 0.14 M to about 0.9M salt, attemperatures ranging from about 20° C. to about 55° C. The presence of ahybridized complex comprising the probe or primer and a complementarypolynucleotide from the test sample indicates the presence of Apl and/oran Apl polynucleotide sequence in the sample.

Antibodies

Antibodies of the invention are antibody molecules that specificallybind to an Anaplasma phagocytophilum polypeptide of the invention,variant polypeptides of the invention, or fragments thereof. An antibodyof the invention can be specific for an Anaplasma phagocytophilumpolypeptide, for example, an antibody specific for one or more of SEQ IDNO: 1, 9 or 11. In another embodiment of the invention an antibody isspecific for both an Anaplasma phagocytophilum polypeptide and anAnaplasma platys polypeptide (e.g., an antibody specific for SEQ IDNO:2, 6, 8, 10 or 12). In another embodiment of the invention anantibody is specific for both an Anaplasma phagocytophilum polypeptideand another Anaplasma polypeptide. One of skill in the art can easilydetermine if an antibody is specific for an Anaplasma phagocytophilumpolypeptide or Anaplasma platys polypeptide using assays describedherein. An antibody of the invention can be a polyclonal antibody, amonoclonal antibody, a single chain antibody (scFv), or an antigenbinding fragment of an antibody. Antigen-binding fragments of antibodiesare a portion of an intact antibody comprising the antigen binding siteor variable region of an intact antibody, wherein the portion is free ofthe constant heavy chain domains of the Fc region of the intactantibody. Examples of antigen binding antibody fragments include Fab,Fab′, Fab′-SH, F(ab′)₂ and F_(v) fragments.

An antibody of the invention can be any antibody class, including forexample, IgG, IgM, IgA, IgD and IgE. An antibody or fragment thereofbinds to an epitope of a polypeptide of the invention. An antibody canbe made in vivo in suitable laboratory animals or in vitro usingrecombinant DNA techniques. Means for preparing and characterizingantibodies are well know in the art. See, e.g., Dean, Methods Mol. Biol.80:23-37 (1998); Dean, Methods Mol. Biol. 32:361-79 (1994); Baileg,Methods Mol. Biol. 32:381-88 (1994); Gullick, Methods Mol. Biol.32:389-99 (1994); Drenckhahn et al. Methods Cell. Biol. 37:7-56 (1993);Morrison, Ann. Rev. Immunol. 10:239-65 (1992); Wright et al. Crit. Rev.Immunol. 12:125-68 (1992). For example, polyclonal antibodies can beproduced by administering a polypeptide of the invention to an animal,such as a human or other primate, mouse, rat, rabbit, guinea pig, goat,pig, dog, cow, sheep, donkey, or horse. Serum from the immunized animalis collected and the antibodies are purified from the plasma by, forexample, precipitation with ammonium sulfate, followed bychromatography, such as affinity chromatography. Techniques forproducing and processing polyclonal antibodies are known in the art.

“Specifically binds,” “specifically bind,” or “specific for” means thata first antigen, e.g., an Anaplasma phagocytophilum polypeptide,recognizes and binds to an antibody of the invention with greateraffinity than to other, non-specific molecules. “Specifically binds,”“specifically bind,” or “specific for” also means a first antibody,e.g., an antibody raised against SEQ ID NOs:1-2, 6, and 8-12, recognizesand binds to SEQ ID NOs:1-2, 6, and 8-12, with greater affinity than toother non-specific molecules. A non-specific molecule is an antigen thatshares no common epitope with the first antigen. In a preferredembodiment of the invention a non-specific molecule is not derived fromAnaplasma sp. An Anaplasma sp. is any species of the genus Anaplasma.For example, an antibody raised against a first antigen (e.g., apolypeptide) to which it binds more efficiently than to a non-specificantigen can be described as specifically binding to the first antigen.In one embodiment, an antibody or antigen-binding portion thereofspecifically binds to a polypeptide of SEQ ID NOs:1, 2, 6, 8, 9, 10, 11,12 or fragments thereof when it binds with a binding affinity K_(a) of10⁷ l/mol or more. Specific binding can be tested using, for example, anenzyme-linked immunosorbant assay (ELISA), a radioimmunoassay (RIA), ora western blot assay using methodology well known in the art.

Antibodies of the invention include antibodies and antigen bindingfragments thereof that (a) compete with a reference antibody for bindingto SEQ ID NOs:1, 2, 6, 8, 9, 10, 11, 12 or antigen binding fragmentsthereof; (b) binds to the same epitope of SEQ ID NOs: 1, 2, 6, 8, 9, 10,11, 12 or antigen binding fragments thereof as a reference antibody; (c)binds to SEQ ID NOs: 1, 2, 6, 8, 9, 10, 11, 12 or antigen bindingfragments thereof with substantially the same K_(d) as a referenceantibody; and/or (d) binds to SEQ ID NOs: 1, 2, 6, 8, 9, 10, 11, 12 orfragments thereof with substantially the same off rate as a referenceantibody, wherein the reference antibody is an antibody orantigen-binding fragment thereof that specifically binds to apolypeptide of SEQ ID NOs: 1, 2, 6, 8, 9, 10, 11, 12 or antigen bindingfragments thereof with a binding affinity K_(a) of 10⁷ l/mol or more.

One embodiment of the invention provides a first purified polypeptidethat specifically binds an antibody, wherein the antibody specificallybinds a second purified polypeptide, wherein the second purifiedpolypeptide consists of SEQ ID NO:1, 2, 6, 8, 9, 10, 11, That is,polypeptides of the invention include polypeptides that can specificallybind to antibodies that are capable of specifically binding to apurified polypeptide that consists of SEQ ID NO:1, 2, 6, 8, 9, 10, 11.The first purified polypeptide can consist of at least 10 but less than300 contiguous amino acids of SEQ ID NO: 1, 2, 6, 8, 9, 10, 11 or 12.The first purified polypeptide can be at least 95% identical to SEQ IDNO: 1, 2, 6, 8, 9, 10, 11 or 12.

Monoclonal antibodies directed against epitopes present on a polypeptideof the invention can also be readily produced. For example, normal Bcells from a mammal, such as a mouse, which was immunized with apolypeptide of the invention can be fused with, for example,HAT-sensitive mouse myeloma cells to produce hybridomas. Hybridomasproducing Anaplasma phagocytophilum-specific antibodies can beidentified using RIA or ELISA and isolated by cloning in semi-solid agaror by limiting dilution. Clones producing Anaplasmaphagocytophilum-specific antibodies are isolated by another round ofscreening. Monoclonal antibodies can be screened for specificity usingstandard techniques, for example, by binding a polypeptide of theinvention to a microtiter plate and measuring binding of the monoclonalantibody by an ELISA assay. Techniques for producing and processingmonoclonal antibodies are known in the art. See e.g., Kohler & Milstein,Nature, 256:495 (1975). Particular isotypes of a monoclonal antibody canbe prepared directly, by selecting from the initial fusion, or preparedsecondarily, from a parental hybridoma secreting a monoclonal antibodyof a different isotype by using a sib selection technique to isolateclass-switch variants. See Steplewski et al., P.N.A.S. U.S.A. 82:86531985; Spria et al., J. Immunolog. Meth. 74:307, 1984. Monoclonalantibodies of the invention can also be recombinant monoclonalantibodies. See, e.g., U.S. Pat. Nos. 4,474,893; 4,816,567. Antibodiesof the invention can also be chemically constructed. See, e.g., U.S.Pat. No. 4,676,980.

Antibodies of the invention can be chimeric (see, e.g., U.S. Pat. No.5,482,856), humanized (see, e.g., Jones et al., Nature 321:522 (1986);Reichmann et al., Nature 332:323 (1988); Presta, Curr. Op. Struct. Biol.2:593 (1992)), caninized, canine, or human antibodies. Human antibodiescan be made by, for example, direct immortilization, phage display,transgenic mice, or a Trimera methodology, see e.g., Reisener et al.,Trends Biotechnol. 16:242-246 (1998).

Antibodies that specifically bind Anaplasma phagocytophilum antigens(e.g., Anaplasma phagocytophilum polypeptides), are particularly usefulfor detecting the presence of Anaplasma phagocytophilum antigens in asample, such as a serum, blood, plasma, urine, fecal, tissue, or salivasample from an Anaplasma phagocytophilum-infected animal. An immunoassayfor Anaplasma phagocytophilum antigen can utilize one antibody orseveral antibodies. An immunoassay for Anaplasma phagocytophilum antigencan use, for example, a monoclonal antibody specific for an Anaplasmaphagocytophilum epitope, a combination of monoclonal antibodies specificfor epitopes of one Anaplasma phagocytophilum polypeptide, monoclonalantibodies specific for epitopes of different Anaplasma phagocytophilumpolypeptides, polyclonal antibodies specific for the same Anaplasmaphagocytophilum antigen, polyclonal antibodies specific for differentAnaplasma phagocytophilum antigens, or a combination of monoclonal andpolyclonal antibodies. Immunoassay protocols can be based upon, forexample, competition, direct reaction, or sandwich type assays using,for example, labeled antibody. Antibodies of the invention can belabeled with any type of label known in the art, including, for example,fluorescent, chemiluminescent, radioactive, enzyme, colloidal metal,radioisotope and bioluminescent labels. Other antibodies of theinvention can specifically bind Aph antigens and Apl antigens, or Aphantigens and other Anaplasma sp. antigens and can be used as describedfor Aph above.

Antibodies of the invention or antigen-binding fragments thereof can bebound to a support and used to detect the presence of Apl, Anaplasma sp.and/or Aph antigens. Supports include, for example, glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agaroses and magletite.

Antibodies of the invention can further be used to isolate Anaplasmasp., Apl, and/or Aph organisms or antigens by immunoaffinity columns.The antibodies can be affixed to a solid support by, for example,adsorbtion or by covalent linkage so that the antibodies retain theirimmunoselective activity. Optionally, spacer groups can be included sothat the antigen binding site of the antibody remains accessible. Theimmobilized antibodies can then be used to bind Anaplasma organisms orAnaplasma antigens from a sample, such as a biological sample includingsaliva, serum, sputum, blood, urine, feces, cerebrospinal fluid,amniotic fluid, wound exudate, or tissue. The bound Anaplasma organismsor Anaplasma antigens are recovered from the column matrix by, forexample, a change in pH.

Antibodies of the invention can also be used in immunolocalizationstudies to analyze the presence and distribution of a polypeptide of theinvention during various cellular events or physiological conditions.Antibodies can also be used to identify molecules involved in passiveimmunization and to identify molecules involved in the biosynthesis ofnon-protein antigens. Identification of such molecules can be useful invaccine development. Antibodies of the invention, including, forexample, monoclonal antibodies and single chain antibodies, can be usedto monitor the course of amelioration of a disease caused by Anaplasmasp., Apl and/or Aph. By measuring the increase or decrease of antibodiesspecific for Apl, Anaplasma sp. and/or Aph in a test sample from ananimal, it can be determined whether a particular therapeutic regimentaimed at ameliorating the disorder is effective. Antibodies can bedetected and/or quantified using for example, direct binding assays suchas RIA, ELISA, or western blot assays.

Methods of Detection

The methods of the invention can be used to detect antibodies orantibody fragments specific for Anaplasma sp., Apl, or Aph; Anaplasmaantigens, Apl antigens, Aph antigens; Anaplasma sp. polynucleotides, Aplpolynucleotides, Aph polynucleotides; or a combination thereof in a testsample, such as a biological sample, an environmental sample, or alaboratory sample. A test sample can potentially comprise Anaplasma sp.polynucleotides, Apl polynucleotides, Aph polynucleotides, Anaplasma sp.polypeptides, Apl polypeptides, Aph polypeptides, antibodies specificfor Anaplasma sp., antibodies specific for Apl, and/or antibodiesspecific for Aph, combinations thereof, unrelated antibodies, unrelatedpolypeptides, unrelated polynucleotides, or none of the above. Abiological sample can include, for example, sera, saliva, blood, cells,plasma, urine, feces, or tissue from a mammal such as a horse, cat, dogor human. The test sample can be untreated, precipitated, fractionated,separated, diluted, concentrated, or purified.

In one embodiment methods of the invention comprise contacting one ormore polypeptides of the invention with a test sample under conditionsthat allow polypeptide/antibody complexes, i.e., immunocomplexes, toform. That is, polypeptides of the invention specifically bind toantibodies specific for Anaplasma sp., Apl and/or Aph antigens locatedin the sample. In one embodiment of the invention one or morepolypeptides of the invention (e.g., SEQ ID NOs:1, 9, 11 or fragmentsthereof) specifically bind to antibodies that are specific for Aphantigens and do not specifically bind to Apl antigens. In anotherembodiment of the invention one or more polypeptides of the invention(e.g., SEQ ID NOs: 2, 6, 8, 10, or 12 or fragments thereof) specificallybind to antibodies that are specific for both Aph and Apl antigens. Oneof skill in the art is familiar with assays and conditions that are usedto detect antibody/polypeptide complex binding. The formation of acomplex between polypeptides and anti-Anaplasma sp., anti-Apl and/oranti-Aph antibodies in the sample is detected.

Antibodies of the invention can be used in a method of the diagnosis ofAnaplasma sp., Apl, and/or Aph infection by obtaining a test samplefrom, e.g., a human or animal suspected of having an Anaplasma sp., Apl,and/or Aph infection. The test sample is contacted with antibodies ofthe invention under conditions enabling the formation ofantibody-antigen complexes (i.e., immunocomplexes). One of skill in theart is aware of conditions that enable and are appropriate for formationof antigen/antibody complexes. The amount of antibody-antigen complexescan be determined by methodology known in the art. A level that ishigher than that formed in a control sample indicates an Anaplasma sp.,Apl, and/or Aph infection. A control sample is a sample that does notcomprise any Aph and/or Apl polypeptides or antibodies specific for Aphand/or Apl. In one embodiment of the invention the control contains noAnaplasma sp. polypeptides or antibodies specific for Anaplasma sp. Inone embodiment of the invention an antibody is specific for Aph antigensonly. In another embodiment of the invention an antibody is specific forboth Aph and Apl antigens. Alternatively, a polypeptide of the inventioncan be contacted with a test sample. Antibodies specific for Anaplasmasp., Apl, and/or Aph in a positive test sample will formantigen-antibody complexes under suitable conditions. The amount ofantibody-antigen complexes can be determined by methods known in theart.

In one embodiment of the invention, Anaplasma phagocytophilum and/orAnaplasma platys infection can be detected in a subject. A biologicalsample is obtained from the subject. One or more purified polypeptidescomprising SEQ ID NO: 2, 6, 8, 10, or 12, are contacted with thebiological sample under conditions that allow polypeptide/antibodycomplexes to form. The purified polypeptides can consist of less thanabout 300 contiguous naturally occurring Anaplasma phagocytophilum aminoacids. The polypeptide/antibody complexes are detected. The detection ofthe polypeptide/antibody complexes is an indication that the mammal hasan Anaplasma phagocytophilum and/or Anaplasma platys infection. BecauseSEQ ID NO: 2, 6, 8, 10, or 12, are specific for both anti-Apl andanti-Aph antibodies, the detected infection can be Aph infection, Aplinfection or both Apl and Aph infection. The lack of detection ofpolypeptide/antibody complexes is an indication that the subject doesnot have an Anaplasma phagocytophilum and/or Anaplasma platys infection.

Another embodiment of the invention provides a method of detecting anAnaplasma phagocytophilum infection in a subject. The method comprisesobtaining a biological sample from the subject and contacting one ormore purified polypeptides comprising SEQ ID NO:1, 9 or 11, with thebiological sample under conditions that allow polypeptide/antibodycomplexes to form. The purified polypeptides can consist of less thanabout 150 contiguous naturally occurring Anaplasma phagocytophilum aminoacids. The polypeptide/antibody complexes are detected. The detection ofthe polypeptide/antibody complexes is an indication that the subject hasan Anaplasma phagocytophilum infection and the lack of detection of thepolypeptide/antibody complexes is an indication that the subject doesnot have an Anaplasma phagocytophilum infection.

In one embodiment of the invention, Apl and/or Aph infection can bedetected in a subject by about 5 days, 6 days, 7 days, 8 days, 9 days,10 days, 12 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days,20 days, 21 days or more after the subject acquired the Apl and/or Aphinfection. In one embodiment of the invention, Apl and/or Aph infectioncan be detected in a subject by about 21 days, 20 days, 19 days, 18days, 17 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10days, 9 days, 8 days, 7 days, 6 days, 5 days, or less after the subjectacquired the Aph and/or Apl infection.

In one embodiment of the invention, the polypeptide/antibody complex isdetected when an indicator reagent, such as an enzyme conjugate, whichis bound to the antibody, catalyzes a detectable reaction. Optionally,an indicator reagent comprising a signal generating compound can beapplied to the polypeptide/antibody complex under conditions that allowformation of a polypeptide/antibody/indicator complex. Thepolypeptide/antibody/indicator complex is detected. Optionally, thepolypeptide or antibody can be labeled with an indicator reagent priorto the formation of a polypeptide/antibody complex. The method canoptionally comprise a positive or negative control.

In one embodiment of the invention, one or more antibodies of theinvention are attached to a solid phase or substrate. A test samplepotentially comprising a protein comprising a polypeptide of theinvention is added to the substrate. One or more antibodies thatspecifically bind polypeptides of the invention are added. Theantibodies can be the same antibodies used on the solid phase or can befrom a different source or species and can be linked to an indicatorreagent, such as an enzyme conjugate. Wash steps can be performed priorto each addition. A chromophore or enzyme substrate is added and coloris allowed to develop. The color reaction is stopped and the color canbe quantified using, for example, a spectrophotometer.

In another embodiment of the invention, one or more antibodies of theinvention are attached to a solid phase or substrate. A test samplepotentially comprising a protein comprising a polypeptide of theinvention is added to the substrate. Second anti-species antibodies thatspecifically bind polypeptides of the invention are added. These secondantibodies are from a different species than the solid phase antibodies.Third anti-species antibodies are added that specifically bind thesecond antibodies and that do not specifically bind the solid phaseantibodies are added. The third antibodies can comprise an indicatorreagent such as an enzyme conjugate. Wash steps can be performed priorto each addition. A chromophore or enzyme substrate is added and coloris allowed to develop. The color reaction is stopped and the color canbe quantified using, for example, a spectrophotometer.

Assays of the invention include, but are not limited to those based oncompetition, direct reaction or sandwich-type assays, including, but notlimited to enzyme linked immunosorbent assay (ELISA), western blot, IFA,radioimmunoassay (RIA), hemagglutination (HA), fluorescence polarizationimmunoassay (FPIA), and microtiter plate assays (any assay done in oneor more wells of a microtiter plate). One assay of the inventioncomprises a reversible flow chromatographic binding assay, for example aSNAP® assay. See U.S. Pat. No. 5,726,010.

Assays can use solid phases or substrates or can be performed byimmunoprecipitation or any other methods that do not utilize solidphases. Where a solid phase or substrate is used, one or morepolypeptides of the invention are directly or indirectly attached to asolid support or a substrate such as a microtiter well, magnetic bead,non-magnetic bead, column, matrix, membrane, fibrous mat composed ofsynthetic or natural fibers (e.g., glass or cellulose-based materials orthermoplastic polymers, such as, polyethylene, polypropylene, orpolyester), sintered structure composed of particulate materials (e.g.,glass or various thermoplastic polymers), or cast membrane film composedof nitrocellulose, nylon, polysulfone or the like (generally syntheticin nature). In one embodiment of the invention a substrate is sintered,fine particles of polyethylene, commonly known as porous polyethylene,for example, 10-15 micron porous polyethylene from Chromex Corporation(Albuquerque, N. Mex.). All of these substrate materials can be used insuitable shapes, such as films, sheets, or plates, or they may be coatedonto or bonded or laminated to appropriate inert carriers, such aspaper, glass, plastic films, or fabrics. Suitable methods forimmobilizing peptides on solid phases include ionic, hydrophobic,covalent interactions and the like.

In one type of assay format, one or more polypeptides can be coated on asolid phase or substrate. A test sample suspected of containing ananti-Anaplasma sp., anti-Apl, and/or anti-Aph antibody orantigen-binding fragment thereof is incubated with an indicator reagentcomprising a signal generating compound conjugated to an antibody orantigen-binding antibody fragment specific for Anaplasma sp., Apl,and/or Aph for a time and under conditions sufficient to formantigen/antibody complexes of either antibodies of the test sample tothe polypeptides of the solid phase or the indicator reagent compoundconjugated to an antibody specific for Anaplasma sp., Apl, and/or Aph tothe polypeptides of the solid phase. The reduction in binding of theindicator reagent conjugated to an anti-Anaplasma sp., anti-Apl, and/oranti-Aph antibody to the solid phase can be quantitatively measured. Ameasurable reduction in the signal compared to the signal generatedfrom, e.g., a confirmed negative Anaplasma sp., Apl, and/or Aph testsample indicates the presence of anti-Anaplasma sp., anti-Apl, and/oranti-Aph antibody in the test sample. This type of assay can quantitatethe amount of anti-Anaplasma sp., anti-Apl, and/or anti-Aph antibodiesin a test sample.

In another type of assay format, one or more polypeptides of theinvention are coated onto a support or substrate. A polypeptide of theinvention is conjugated to an indicator reagent and added to a testsample. This mixture is applied to the support or substrate. Ifantibodies specific for Anaplasma sp., Apl, and/or Aph are present inthe test sample they will bind the one or more polypeptides conjugatedto an indicator reagent and to the one or more polypeptides immobilizedon the support. The polypeptide/antibody/indicator complex can then bedetected. This type of assay can quantitate the amount of anti-Anaplasmasp., anti-Apl, and/or anti-Aph antibodies in a test sample.

In another type of assay format, one or more polypeptides of theinvention are coated onto a support or substrate. The test sample isapplied to the support or substrate and incubated. Unbound componentsfrom the sample are washed away by washing the solid support with a washsolution. If Anaplasma sp. specific, Apl specific, and/or Aph specificantibodies are present in the test sample, they will bind to thepolypeptide coated on the solid phase. This polypeptide/antibody complexcan be detected using a second species-specific antibody that isconjugated to an indicator reagent. Thepolypeptide/antibody/anti-species antibody indicator complex can then bedetected. This type of assay can quantitate the amount of anti-Anaplasmasp., anti-Apl, and/or anti-Aph antibodies in a test sample.

The formation of a polypeptide/antibody complex or apolypeptide/antibody/indicator complex can be detected by e.g.,radiometric, colorimetric, fluorometric, size-separation, orprecipitation methods. Optionally, detection of a polypeptide/antibodycomplex is by the addition of a secondary antibody that is coupled to anindicator reagent comprising a signal generating compound. Indicatorreagents comprising signal generating compounds (labels) associated witha polypeptide/antibody complex can be detected using the methodsdescribed above and include chromogenic agents, catalysts such as enzymeconjugates fluorescent compounds such as fluorescein and rhodamine,chemiluminescent compounds such as dioxetanes, acridiniums,phenanthridiniums, ruthenium, and luminol, radioactive elements, directvisual labels, as well as cofactors, inhibitors, magnetic particles, andthe like. Examples of enzyme conjugates include alkaline phosphatase,horseradish peroxidase, beta-galactosidase, and the like. The selectionof a particular label is not critical, but it will be capable ofproducing a signal either by itself or in conjunction with one or moreadditional substances.

Formation of the complex is indicative of the presence of anti-Anaplasmasp., anti-Apl, and/or anti-Aph antibodies in a test sample. Therefore,the methods of the invention can be used to diagnose Anaplasma sp., Apl,and/or Aph infection in a patient.

The methods of the invention can also indicate the amount or quantity ofanti-Anaplasma sp., anti-Apl and/or anti-Aph antibodies in a testsample. With many indicator reagents, such as enzyme conjugates, theamount of antibody present is proportional to the signal generated.Depending upon the type of test sample, it can be diluted with asuitable buffer reagent, concentrated, or contacted with a solid phasewithout any manipulation. For example, it usually is preferred to testserum or plasma samples that previously have been diluted, orconcentrated specimens such as urine, in order to determine the presenceand/or amount of antibody present.

The invention further comprises assay kits (e.g., articles ofmanufacture) for detecting anti-Anaplasma sp., anti-Apl and/or anti-Aphantibodies or antigen-binding antibody fragments, Anaplasmapolypeptides, Apl polypeptides, and/or Aph polypeptides in a sample. Akit comprises one or more polypeptides of the invention and means fordetermining binding of the polypeptide to anti-Anaplasma sp. antibodies,anti-Apl antibodies, and/or or anti-Aph antibodies or antigen-bindingantibody fragments in the sample. A kit or article of manufacture canalso comprise one or more antibodies or antigen-binding antibodyfragments of the invention and means for determining binding of theantibodies or antigen-binding antibody fragments to Anaplasma sp.polypeptides, Apl polypeptides, and/or Aph polypeptides in the sample. Akit can comprise a device containing one or more polypeptides orantibodies of the invention and instructions for use of the one or morepolypeptides or antibodies for, e.g., the identification of an Anaplasmasp., Apl, and/or Aph infection in a mammal. The kit can also comprisepackaging material comprising a label that indicates that the one ormore polypeptides or antibodies of the kit can be used for theidentification of Anaplasma sp., Apl, and/or Aph infection. Othercomponents such as buffers, controls, and the like, known to those ofordinary skill in art, can be included in such test kits. Thepolypeptides, antibodies, assays, and kits of the invention are useful,for example, in the diagnosis of individual cases of Anaplasma sp., Apl,and/or Aph infection in a patient, as well as epidemiological studies ofAnaplasma sp., Apl, and/or Aph outbreaks.

Polypeptides and assays of the invention can be combined with otherpolypeptides or assays to detect the presence of Anaplasma sp. alongwith other organisms. For example, polypeptides and assays of theinvention can be combined with reagents that detect heartworm and/orBorrelia burgdorferi and/or Ehrlichia canis.

Polynucleotides of the invention can be used to detect the presence ofAnaplasma sp., Apl and/or Aph polynucleotides in a sample. Thepolynucleotides can be used to detect Anaplasma sp., Apl and/or Aphpolynucleotides in a sample by a simple hybridization reaction and canalso be used in, e.g., polymerase chain reactions (PCR) such as areal-time PCR reaction. Methods and compositions of the invention canalso be used to differentially detect the presence Aph from otherAnaplasma sp., such as Apl.

PCR assays are well described in the art, including, for example, U.S.Pat. Nos. 4,683,195; 4,683,202; 4,965,188. Generally, polynucleotideprimers are annealed to denatured strands of a target nucleic acid.Primer extension products are formed by polymerization ofdeoxynucleoside triphosphates by a polymerase. PCR then involvesrepetitive cycles of template nucleic acid denaturation, primerannealing and extension of the annealed primers by the action of athermostable polymerase. The process results in exponentialamplification of the target Anaplasma sp. nucleic acids in the testsample, which allows for the detection of target polynucleotidesexisting in very low concentrations in a sample.

Real-time PCR assays are based on the detection of a signal, e.g., afluorescent reporter signal. This signal increases in direct proportionto the amount of PCR product in a reaction. Real-time PCR is anyamplification technique that makes it possible to monitor the evolutionof an ongoing amplification reaction. See, Quantitation of DNA/RNA UsingReal-Time PCR Detection, Perkin Elmer Applied Biosystems (1999); PCRProtocols (Academic Press New York, 1989). By recording the amount offluorescence emission at each cycle, it is possible to monitor the PCRreaction during exponential phase where the first significant increasein the amount of PCR product correlates to the initial amount of targettemplate. The higher the starting copy number of the nucleic acidtarget, the sooner a significant increase in fluorescence is observed.

One embodiment of the invention provides a method for detecting and/orquantifying Anaplasma sp., Apl and/or Aph polynucleotides in a testsample. Sense primers and antisense primers can be added to a testsample under conditions suitable for a polymerase chain reaction. Theprimers hybridize with Anaplasma sp., Apl and/or Aph polynucleotidessuch that an amplification product is formed if Anaplasma sp., Apland/or Aph polynucleotides are present in the test sample. Amplificationproducts are detected and the presence and/or quantity of Anaplasma sp.,Apl and/or Aph polynucleotides is determined. Amplification products canbe detected with a polynucleotide probe that hybridizes, underconditions suitable for a polymerase chain reaction, with an Anaplasmasp., Apl and/or Aph polynucleotide sequence. The amplification productcan be quantified by measuring a detection signal from the probe andcomparing said detection signal to a second probe detection signal froma quantification standard. The quantification standard can be extractedin parallel with the test sample.

One embodiment of the invention provides a method for differentiallydetecting Anaplasma platys polypeptides and Anaplasma phagocytophilumpolypeptides in a sample. The method comprises:

-   -   (a) contacting one or more antibodies that specifically bind to        a polypeptide consisting of SEQ ID NO: 2, 6, 8, 10, or 12 with a        sample under conditions that allow polypeptide/antibody        complexes to form and detecting the polypeptide/antibody        complexes; and    -   (b) contacting one or more antibodies that specifically bind to        a polypeptide consisting of SEQ ID NO:1, 9, or 11 with the        sample under conditions that allow polypeptide/antibody        complexes to form and detecting the polypeptide/antibody        complexes.

If the polypeptide/antibody complexes are detected in step (a) and instep (b) then the sample contains Anaplasma phagocytophilum polypeptidesand may also contain Anaplasma platys polypeptides. Another test can berun that is specific for Anaplasma platys, if desired, in order todetermine if Anaplasma platys is indeed present. If thepolypeptide/antibody complexes are detected in step (a) and are notdetected in step (b) then the sample contains Anaplasma platyspolypeptides and does not contain Anaplasma phagocytophilumpolypeptides. If the polypeptide complexes are not detected in step (a)and are not detected in step (b) then the sample does not containAnaplasma platys polypeptides and does not contain Anaplasmaphagocytophilum polypeptides.

Another embodiment of the invention provides a method of detectingantibodies that specifically bind an Anaplasma platys polypeptide, anAnaplasma phagocytophilum polypeptide, or both an Anaplasma platyspolypeptide and an Anaplasma phagocytophilum polypeptide. The methodcomprises:

-   -   (a) contacting one or more purified polypeptides comprising SEQ        ID NO: 2, 6, 8, 10, or 12, with a test sample, under conditions        that allow polypeptide/antibody complexes to form and detecting        the polypeptide/antibody complexes; and    -   (b) contacting one or more purified polypeptides comprising SEQ        ID NO:1, 9, or 11, wherein the purified polypeptide with a test        sample, under conditions that allow polypeptide/antibody        complexes to form and detecting the polypeptide/antibody        complexes.

If the polypeptide/antibody complexes are detected in step (a) and instep (b) then the sample contains antibodies the specifically bindAnaplasma phagocytophilum polypeptides and Anaplasma platys polypeptides(that is, antibodies that are capable of specifically binding bothAnaplasma platys and Anaplasma phagocytophilum polypeptides) andantibodies that specifically bind only Anaplasma platys polypeptides. Ifthe polypeptide/antibody complexes are detected in step (a) and are notdetected in step (b) then the sample contains antibodies thatspecifically bind Anaplasma platys polypeptides and Anaplasmaphagocytophilum polypeptides and does not contain antibodies thatspecifically bind only Anaplasma phagocytophilum polypeptides. If thepolypeptide complexes are not detected in step (a) and are not detectedin step (b) then the sample does not contain antibodies specific forAnaplasma platys polypeptides and does not contain antibodies specificfor Anaplasma phagocytophilum polypeptides. In one embodiment, thepurified polypeptides comprising SEQ ID NO: 2, 6, 8, 10, or 12, consistof less than about 300 contiguous naturally occurring Anaplasmaphagocytophilum amino acids and the polypeptides comprising SEQ ID NO:1,9, or 11, consist of less than about 150 contiguous naturally occurringAnaplasma phagocytophilum amino acids.

Methods of Treatment, Amelioration, or Prevention of a Disease Caused byAph and/or Apl

Polypeptides, polynucleotides, and antibodies of the invention can beused to treat, ameliorate, or prevent a disease caused by Apl and/orAph. For example, an antibody, such as a monoclonal antibody of theinvention or antigen-binding fragments thereof, can be administered toan animal, such as a human or dog. In one embodiment of the invention anantibody or antigen-binding fragment thereof is administered to ananimal in a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier. A pharmaceutical composition comprises atherapeutically effective amount of an antibody or antigen-bindingfragments thereof. A therapeutically effective amount is an amounteffective in alleviating the symptoms of an Apl and/or Aph infection orin reducing the amount of Apl and/or Aph organisms in a subject.

Polypeptides or polynucleotides of the invention can be present in animmunogenic composition and used to elicit an immune response in a host.An immunogenic composition or immunogen is capable of inducing an immuneresponse in an animal. An immunogenic polypeptide or polynucleotidecomposition of the invention is particularly useful in sensitizing animmune system of an animal such that, as one result, an immune responseis produced that ameliorates or prevents the effect of Apl and/or Aphinfection. The elicitation of an immune response in animal model can beuseful to determine, for example, optimal doses or administrationroutes. Elicitation of an immune response can also be used to treat,prevent, or ameliorate a disease or infection caused by Apl and/or Aph.An immune response includes humoral immune responses or cell mediatedimmune responses, or a combination thereof. An immune response can alsocomprise the promotion of a generalized host response, e.g., bypromoting the production of defensins.

One embodiment of the invention provides an immunogen that comprises apolypeptide of the invention and one or more additional regions ormoieties covalently joined to the polypeptide at the carboxyl terminusor amino terminus. Each region or moiety can, for example, enhance theimmune response, facilitate purification of the immunogen, or facilitatepolypeptide stability.

Polypeptide stability can be enhanced by adding, for example,polyethylene glycol to the amino or carboxyl terminus of thepolypeptide. Other regions or moieties that can improve polypeptidestability include, for example, amino terminus protecting groups such asacetyl, propyl, succinyl, benzyl, benzyloxycarbonyl ort-butyloxycarbonyl and carboxyl terminus protecting groups such asamide, methylamide, and ethylamide.

Polypeptide purification can be enhanced by adding a region or moiety tothe carboxyl or amino terminus to facilitate purification. Examples ofgroups that can be used to facilitate purification include affinity tagssuch as six-histidine tag, trpE, glutathione and maltose-bindingprotein.

The ability of a polypeptide to produce an immune response can beenhanced with certain additional regions or moieties. Examples of groupsthat can be joined to a polypeptide to enhance an immune responseinclude cytokines such as IL-2. See, e.g., Buchan et al., 2000.Molecular Immunology 37:545-552.

The generation of an antibody titer by an animal against Apl and/or Aphcan be important in protection from infection and clearance ofinfection. Detection and/or quantification of antibody titers afterdelivery of a polypeptide or polynucleotide can be used to identifyepitopes that are particularly effective at eliciting antibody titers.Epitopes responsible for a strong antibody response to Apl and/or Aphcan be identified by eliciting antibodies directed against Apl and/orAph polypeptides of different lengths. Antibodies elicited by aparticular polypeptide epitope can then be tested using, for example, anELISA assay to determine which polypeptides contain epitopes that aremost effective at generating a strong response. Polypeptides or fusionproteins that contain these epitopes or polynucleotides encoding theepitopes can then be constructed and used to elicit a strong antibodyresponse.

A polypeptide, polynucleotide, or antibody of the invention can beadministered to a mammal, such as a mouse, rabbit, guinea pig, macaque,baboon, chimpanzee, human, cow, sheep, pig, horse, dog, cat, or toanimals such as chickens or ducks, to elicit antibodies in vivo.Injection of a polynucleotide has the practical advantages of simplicityof construction and modification. Further, injection of a polynucleotideresults in the synthesis of a polypeptide in the host. Thus, thepolypeptide is presented to the host immune system with nativepost-translational modifications, structure, and conformation. Apolynucleotide can be delivered to a subject as “naked DNA.”

Administration of a polynucleotide, polypeptide, or antibody can be byany means known in the art, including intramuscular, intravenous,intrapulmonary, intramuscular, intradermal, intraperitoneal, orsubcutaneous injection, aerosol, intranasal, infusion pump, suppository,mucosal, topical, and oral, including injection using a biologicalballistic gun (“gene gun”). A polynucleotide, polypeptide, or antibodycan be accompanied by a protein carrier for oral administration. Acombination of administration methods can also be used to elicit animmune response. Antibodies can be administered at a daily dose of about0.5 mg to about 200 mg. In one embodiment of the invention antibodiesare administered at a daily dose of about 20 to about 100 mg.

Pharmaceutically acceptable carriers and diluents and veterinarilyacceptable carries and diluents for therapeutic use are well known inthe art and are described in, for example, Remington's PharmaceuticalSciences, Mack Publishing Co. (A.R. Gennaro ed. (1985)). The carriershould not itself induce the production of antibodies harmful to thehost. Such carriers include, but are not limited to, large, slowlymetabolized, macromolecules, such as proteins, polysaccharides such aslatex functionalized SEPHAROSE®, agarose, cellulose, cellulose beads andthe like, polylactic acids, polyglycolic acids, polymeric amino acidssuch as polyglutamic acid, polylysine, and the like, amino acidcopolymers, peptoids, lipitoids, and inactive, avirulent virus particlesor bacterial cells. Liposomes, hydrogels, cyclodextrins, biodegradablenanocapsules, and bioadhesives can also be used as a carrier for acomposition of the invention.

Pharmaceutically acceptable salts can also be used in compositions ofthe invention, for example, mineral salts such as hydrochlorides,hydrobromides, phosphates, or sulfates, as well as salts of organicacids such as acetates, proprionates, malonates, or benzoates.Especially useful protein substrates are serum albumins, keyhole limpethemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanustoxoid, and other proteins well known to those of skill in the art.Compositions of the invention can also contain liquids or excipients,such as water, saline, phosphate buffered saline, Ringer's solution,Hank's solution, glucose, glycerol, dextrose, malodextrin, ethanol, orthe like, singly or in combination, as well as substances such aswetting agents, emulsifying agents, tonicity adjusting agents,detergent, or pH buffering agents. Additional active agents, such asbacteriocidal agents can also be used.

If desired, co-stimulatory molecules, which improve immunogenpresentation to lymphocytes, such as B7-1 or B7-2, or cytokines such asMIP1a, GM-CSF, IL-2, and IL-12, can be included in a composition of theinvention. Optionally, adjuvants can also be included in a composition.Adjuvants are substances that can be used to nonspecifically augment aspecific immune response. Generally, an adjuvant and a polypeptide ofthe invention are mixed prior to presentation to the immune system, orpresented separately, but are presented into the same site of theanimal. Adjuvants can include, for example, oil adjuvants (e.g. Freund'scomplete and incomplete adjuvants) mineral salts (e.g. Alk(SO₄)₂;AlNa(SO₄)₂, AlNH₄(SO₄), Silica, Alum, Al(OH)₃, and Ca₃(PO₄)₂),polynucleotides (i.e. Poly IC and Poly AU acids), and certain naturalsubstances (e.g. wax D from Mycobacterium tuberculosis, as well assubstances found in Corynebacterium parvum, Bordetella pertussis andmembers of the genus Brucella. Adjuvants which can be used include, butare not limited to MF59-0, aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637), referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/TWEEN®(polysorbate) 80 emulsion.

The compositions of the invention can be formulated into ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, injectable formulations, mouthwashes, dentrifices, andthe like. The percentage of one or more polypeptides, polynucleotides,or antibodies of the invention in such compositions and preparations canvary from 0.1% to 60% of the weight of the unit.

Administration of polypeptides, polynucleotides, or antibodies canelicit an immune response in the animal that lasts for at least 1 week,1 month, 3 months, 6 months, 1 year, or longer. Optionally, an immuneresponse can be maintained in an animal by providing one or more boosterinjections of the polypeptide, polynucleotide, or antibodies at 1 month,3 months, 6 months, 1 year, or more after the primary injection. Ifdesired, co-stimulatory molecules or adjuvants can also be providedbefore, after, or together with the compositions.

A composition of the invention comprising a polypeptide, polynucleotide,antibody, or a combination thereof is administered in a mannercompatible with the particular composition used and in an amount that iseffective to elicit an immune response as detected by, for example, anELISA. A polynucleotide can be injected intramuscularly to a mammal,such as a baboon, chimpanzee, dog, or human, at a dose of 1 ng/kg, 10ng/kg, 100 ng/kg, 1000 ng/kg, 0.001 mg/kg, 0.1 mg/kg, or 0.5 mg/kg. Apolypeptide or antibody can be injected intramuscularly to a mammal at adose of 0.01, 0.05, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 5 or 10 mg/kg.

Polypeptides, polynucleotides, or antibodies, or a combination thereofcan be administered either to an animal that is not infected with Apland/or Aph or can be administered to an Apl and/or Aph-infected animal.An immunologically effective amount or therapeutically effective amountmeans the administration of that amount to an individual, either in asingle dose or as part of series, is effective for treatment,amelioration, or prevention of Apl and or Aph infection. The particulardosages of polynucleotide, polypeptides, or antibodies in a compositionwill depend on many factors including, but not limited to the species,age, gender, concurrent medication, general condition of the mammal towhich the composition is administered, and the mode of administration ofthe composition. An effective amount of the composition of the inventioncan be readily determined using only routine experimentation.

All patents, patent applications, and other scientific or technicalwritings referred to anywhere herein are incorporated by reference intheir entirety. The invention illustratively described herein suitablycan be practiced in the absence of any element or elements, limitationor limitations that are not specifically disclosed herein. Thus, forexample, in each instance herein any of the terms “comprising”,“consisting essentially of”, and “consisting of” may be replaced witheither of the other two terms, while retaining their ordinary meanings.The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by embodiments,optional features, modification and variation of the concepts hereindisclosed may be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the description and the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

EXAMPLES Example 1 Sera from Aph or Apl Infected Dogs React Specificallywith One or More Antigens from a Hypothetical Aph Open Reading Frame

Based on a combination of genomic and protein structural analysis, thehypothetical open reading frame (ORF) Aph 0915 was selected from thegenome of Aph (Dunning Hotopp et al. PLoS Genet. 2(2): e21 (February2006)). Two polynucleotide regions (SEQ ID NOs: 3 and 4) encodingC-terminal fragments of the APH_(—)0915 ORF were PCR amplified usinggenomic Aph DNA isolated from a blood sample of a Minnesota dog known tobe infected with Aph. The PCR products were cloned into an expressionvector for protein expression in E. coli according to methods well knownin the art. Following induction of recombinant protein expression, crudeE. coli whole cell lysates were analyzed for expression of therecombinant protein, and for reactivity to sera from Aph or Apl infecteddogs, using SDS-PAGE electrophoresis and western blotting according tomethods well known in the art. The dog sera were obtained fromexperimentally infected animals, and were used at 1:300 dilution. Crudelysates from E. coli vectors comprising SEQ ID NO:3 or SEQ ID NO:4,expressing polypeptides of SEQ ID NO: 2 (wherein the X at position 43was A; wherein the X at position 113 was D; wherein the X at position133 was E; and wherein the X at position 138 was absent) or SEQ ID NO:1(wherein the X at position 52 was D; the X at position 72 was E; and theX at position 77 was absent), respectively, exhibited specific antigenreactivity (above background levels of E. coli cross reactivity) to serafrom Aph infected dogs. In addition, the crude lysate from clone 13(expressing polypeptide of SEQ ID NO: 2, wherein the X at position 43was A; wherein the X at position 113 was D; wherein the X at position133 was E; and wherein the X at position 138 was absent) exhibitedspecific antigen reactivity to sera from Apl infected dogs. However,crude lysate from clone 14, expressing polypeptide of SEQ ID NO:1(wherein the X at position 52 was D; the X at position 72 was E; and theX at position 77 was absent) did not exhibit specific antigen reactivityto sera from Apl infected dogs.

In addition, the full-length APH 0915 ORF (SEQ ID NO:5, having a G atposition 667, an A at position 878, an A at position 938, and wherein953, 954, and 955 are absent) encoding the full-length APH_(—)0915protein (SEQ ID NO:6 having an A at position 223, a D at position 293,an E at position 313, and no amino acid at position 318), and apolynucleotide region termed “clone 13ext” (SEQ ID NO:7; wherein the Nat position 187 was G; wherein the N at position 398 was A; wherein theN at position 458 was A; and wherein the three N's at positions 473-475were absent) encoding a C-terminal fragment (SEQ ID NO:8; wherein the Xat position 63 was A; wherein the X at position 133 was D; wherein the Xat position 153 was E; and wherein the X at position 158 was absent) ofAPH_(—)0915 were PCR cloned and expressed in E. coli as described abovein this Example. The resulting crude whole cell lysates exhibitedspecific antigen reactivity to dog serum (from a naturally Aph infecteddog exhibiting clinical symptoms of Anaplasmosis) on western blot. Thus,polypeptides comprising SEQ ID NOs:1, 2, 6 and 8 reacted with antibodiesfrom sera of Aph infected dogs. The polypeptide having SEQ ID NO:1 didnot react with antibodies from Apl infected dogs, and was thereforespecific for Aph only.

Example 2 Nucleotide Sequence and Comparison of Clones 13 and 14

The DNA sequence of clones 13 and 14 were determined. The sequences arealigned with the sequence of APH_(—)0915 (Genbank Acc. No. ABD43857)below. (SEQ ID NOs 5, 4, and 3).

APH_0915 1 TTGAGTTTTACAATGTCGAAGTTATCGCTTGACCCTACTCAGGGCTCACATACAGCAGAG61 AATATTGCTTGTTCTATCTTTGATATGGTACTTGGTGTAAAGTCCACTGCAAAACTGTTA 121GCAGGTACGTGGGCTGGTACAAGCAGCACTATTTGGAAGACAGTAACAGGAGCAGCTTCC 181TCAACTAAAGAAGCGTCATCAAAGTCGTATGGAACCCTACGTAGTTCCTTGGGCTCTTCC 241GCTTCTAGAAGGATGCTAGGAACTTGCGCTACCGCCGCTCTCTGCTTAACTGCACCTTTG 301CTTGGCGCAGCCGCTGCCGGAGCGGCAATAACATGTGCCTTGATAACCATTTGCATGGCT 361TTGCTGTTCCTCGTTTTGTACACCGTACTCCACATTGCCTCTCAGATGTTGCGTTGTGCA 421TCGCTACTGTTGAGCATGGTATGCAATATCCTGCACAGCACATTCACCGCAACTAAGTCT 481TGCCTCGGAGGTAAGTCACCTGCGCGAACAACTGAAGAGCGGGTAGCTGGGGATTTAGAT APH_0915541 CACAAAGGGGTGGATTCAGATCGGAAGCATGATGCAGAGAAAACAGAAGAGAAAAAACAT clone131 CACAAAGGGGTGGATTCAGATCGGAAGCATGATGCAGAGAAAACAGAAGAGAAAAAACAT APH_0915601 GGTTTGGGTAGCCTCTGCAAATCACTCGCGATAAATCTGGTCTCCTTAATGGGAACAGCG clone1361 GGTTTGGGTAGCCTCTGCAAATCACTCGCGATAAATCTGGTCTCCTTAATGGGAACAGCG APH_0915661 CTAGTTACCACACCCATAATACTACTTGCAGTAGTTCTATTAGTGTTGGTGCCAGTATAT clone13121 CTAGTTGCCACACCCATAATACTACTTGCAGTAGTTCTATTAGTGTTGGTGCCAGTATATAPH_0915 721CTGTTATGCGCTACAGTGCACCACATCTATCAAGGAAATTACGAAGATCGCAACAACGAC clone13 181CTGTTATGCGCTACAGTGCACCACATCTATCAAGGAAATTACGAAGATCGCAACAACGAC clone14 1---TTATGCGCTACAGTGCACCACATCTATCAAGGAAATTACGAAGATCGCAACAACGAC APH_0915781 AAAGGTAGCTCCCGTGGCGGCGGTACTACATATTATCCAATGACAATGTCTGCAAGTGCT clone13241 AAAGGTAGCTCCCGTGGCGGCGGTACTACATATTATCCAATGACAATGTCTGCAAGTGCT clone1458 AAAGGTAGCTCCCGTGGCGGCGGTACTACATATTATCCAATGACAATGTCTGCAAGTGCT APH_0915841 TCTGAAGAGTCCCTTAGCAGCATAATATCTGAAGGAGGTTTGAGTAAGACATCGCTACCA clone13301 TCTGAAGAGTCCCTTAGCAGCATAATATCTGAAGGAGATTTGAGTAAGACATCGCTACCA clone14118 TCTGAAGAGTCCCTTAGCAGCATAATATCTGAAGGAGATTTGAGTAAGACATCGCTACCAAPH_0915 901AGTTACTCCGCAGCCACTGCTACAGGTACTGGAAATGCAACTGGTGAGGTTTTTTCACAT clone13 361AGTTACTCCGCAGCCACTGCTACAGGTACTGGAAATGAAACTGGTGAGGTTT---CACAT clone14 178AGTTACTCCGCAGCCACTGCTACAGGTACTGGAAATGAAACTGGTGAGGTTT---CACAT APH_0915961 TCTCATTCATCTGGTAAAAGTAGCAGCAAACCAGAATCTCGCCCTGAGAGCAATCTACAG clone13418 TCTCATTCATCTGGTAAAAGTAGCAGCAAACCAGAATCTCGCCCTGAGAGCAATCTACAG clone14235 TCTCATTCATCTGGTAAAAGTAGCAGCAAACCAGAATCTCGCCCTGAGAGCAATCTACAGAPH_0915 1021 AATGTGGTAGCAGAAACCATGTCGCAGCAACAAAGGAGCGTCTCC(SEQ ID NO: 5) clone13 478 AATGTGGTAGCAGAAACCATGTCGCAGCAACAAAGGAGCGTCTCC(SEQ ID NO: 4) clone14 295 AATGTGGTAGCAGAAACCATGTCGCAGCAACAAAGGAGCGTCTCC(SEQ ID NO: 3)The underlining represents differences between the clone 13/clone 14sequences and the Aph_(—)0915 genomic sequence. Furthermore, the DNAsequence of clone 13ext and that of a full-length clone were determined,revealing the same sequence differences (as compared to Genbank Acc. No.ABD43857) identified in clone 13 and clone 14.

Example 3 Clone 13, but not Clone 14, Detects Apl in Field Samples

An experiment was conducted to determine the reactivity of SEQ ID NO:1(clone 14) (wherein the X at position 52 was D; the X at position 72 wasE; and the X at position 77 was absent) or SEQ ID NO:2 (clone 13)(wherein the X at position 43 was A; wherein the X at position 113 wasD; wherein the X at position 133 was E; and wherein the X at position138 was absent) with sera from Apl-infected dogs on western blots (Table2). Sera from thirteen dogs known to be infected with Apl were tested.These sera were derived from dogs living on the Hopi IndianReservations, Arizona, where only Apl (not Aph) is prevalent. See, Table2.

SEQ ID NO:1 (wherein the X at position 52 was D; the X at position 72was E; and the X at position 77 was absent) was not reactive with any ofthe 13 Apl positive samples. Thus, SEQ ID NO:1 is specific for anti-Aphantibodies (see, Example 1) in that it does not react with anti-Aplantibodies.

SEQ ID NO:2 (wherein the X at position 43 was A; wherein the X atposition 113 was D; wherein the X at position 133 was E; and wherein theX at position 138 was absent) was reactive with all 13 of the Aplpositive samples. Thus, SEQ ID NO:2 can detect both anti-Aph (see,examples 1 and 5) and anti-Apl antibodies. See, Table 2.

TABLE 2 Hopi Apl clone 14 clone 13 Sample western western HP 118 − + HP127 − + HP 136 − + HP 141 − + HP 166 − + HP 168 − + HP 170 − + HP 186− + HP 199 − + HP 214 − + HP 230 − + HP 235 − + HP 242 − +

Example 4 Detection of Aph by Clone 14 with Field Samples

Fifty canine serum samples from an area of Minnesota where only Aph (notApl) is prevalent, were tested in this experiment. Of these 50 samples,47 were known Aph positives, and 3 samples were known Aph negatives.Sensitivity of SEQ ID NO:1 (clone 14; wherein the X at position 52 wasD; the X at position 72 was E; and the X at position 77 was absent), forAph was tested by western blots (as described above). Out of the 47known positive samples, 39 were positive on the clone 14 western assay.Out of the 3 known negative samples, 2 were negative, and 1 positive, onthe clone 14 western assay (Table 3). Thus, the SEQ ID NO:1 assay showed83% (39/47) sensitivity for Aph positive samples (Table 3).

TABLE 3 Clone 14 western assay Positive Negative Aph Positive 39 8 AphNegative 1 2

Example 5 Detection of Aph Antibody by SEQ ID NO:2 (Clone 13)

An experiment was conducted to determine whether a polypeptidecomprising SEQ ID NO:2 (clone 13) (wherein the X at position 43 was A;wherein the X at position 113 was D; wherein the X at position 133 wasE; and wherein the X at position 138 was absent) can detect Aphantibodies in sera from dogs exhibiting clinical symptoms of acuteAnaplasmosis.

A purified preparation of a recombinant polypeptide comprising SEQ IDNO:2 (wherein the X at position 43 was A; wherein the X at position 113was D; wherein the X at position 133 was E; and wherein the X atposition 138 was absent) was coated on Immulon® 4 wells overnight at 3ug/mL in carbonate buffer, pH 9.6. The wells were washed 3× with PBSTand blocked with 1% BSA in PBS for 1 h. Test samples (dog sera) diluted1:200 in PRRS Sample Diluent, were added to the wells and incubated for1 h. The wells were washed 6× with HW PetChek® Wash Buffer. Anti-dog(H+L):HRPO 1:4000 in Enzyme Conjugate Diluent was added and incubatedfor 1 h. The wells were washed 6× with HW PetChek® Wash Buffer. 50 μlTMB was added for 10 min and then of 50 μl stop solution was added. Thewells were read at A650.

This indirect ELISA was performed with sera from Aph infected dogsexhibiting clinical symptoms of acute anaplasmosis. Four random samplesfrom normal dogs were tested as negative controls to determine a cut offOD value (cut off=mean+2×SD). Test samples producing OD values abovethis cutoff value were deemed “positives” and are represented by “+” inTable 4.

TABLE 4 ID Clone 13 ME-485 + ME-487 + ME-560 + ME-562 + ME-568 +ME-601 + ME-616 + ME-631 + ME-661 + ME-670 + ME-699 + ME-703 + ME-710 +ME-716 + ME-734 + ME-741 + ME-748 − ME-753 + ME-476 + ME-534 + ME-630 +ME-639 − ME-653 − ME-691 − ME-700 + ME-746 +

Of the 26 samples tested, 22 samples were positive in the ELISA usingSEQ ID NO:2. These results demonstrate that a polypeptide comprising SEQID NO:2 can detect Aph antibodies in sera from dogs having symptoms ofacute anaplasmosis with high sensitivity.

Example 6 Detection of Aph Antibody with Synthetic Polypeptides

Synthetic polypeptides having an amino acid sequence of SEQ ID NO:9, SEQID NO:10, or SEQ ID NO:11 were evaluated for their ability to detect Aphantibody in an indirect ELISA assay. Each of the three peptides wascoated on microtiter plates in serial dilution (0.0-0.5 ul/ml) andtested for reactivity to serum obtained from a naturally infected dog(this dog exhibited symptoms of acute anaplasmosis, and was a knownseropositive for Aph infection). A pooled sample from 10 normal dogs wasused as negative control.

Briefly, synthetic polypeptides having amino acid sequences of SEQ IDNO:9 (p37-1), SEQ ID NO:10 (p37-2), or SEQ ID NO:11 (p3′7-3; wherein theX at position 1 was C; wherein the X at position 2 was E; and whereinthe X at position 7 was absent), were coated onto Immulon® 4 wellsovernight in carbonate buffer, pH 9.6. The wells were washed 2× with HWPetChek® Wash Buffer, followed by blocking for 2 hours with 2% TWEEN®(polysorbate) 20, 2.5% Sucrose in 0.1M Tris (pH 7.6), and driedovernight. Dog serum diluted (1:100) in IBR Conjugate Diluent (IDEXXLaboratories) was added to the wells and incubated for 45 min. The wellswere washed 5× with HW PetChek® Wash Buffer. Rabbit anti-dog antibody(H+L) conjugated to HRPO diluted (1:2000) in IBR Conjugate Diluent wasadded and incubated for 45 min. The wells were washed 6× with HWPetChek® Wash Buffer. 60 μl TMB was added for 10 min and then of 50 μlstop solution was added. The optical density (OD) was read at A650(Table 5).

TABLE 5 Aph normal peptide dog dog concentration serum serum peptide(ug/ml) (OD) (OD) p37-1 0 0.28 0.15 0.06 0.56 0.15 0.125 0.59 0.16 0.250.58 0.18 0.5 0.72 0.19 p37-2 0 0.30 0.17 0.06 0.48 0.16 0.125 1.06 0.160.25 1.93 0.19 0.5 2.28 0.22 p37-3 0 0.30 0.15 0.06 0.45 0.15 0.125 0.560.15 0.25 0.82 0.19 0.5 1.26 0.37

The results in Table 5 show that all three synthetic polypeptides tested(SEQ ID NO:9 (p37-1), SEQ ID NO:10 (p37-2), SEQ ID NO:11 (p3′7-3)(wherein the X at position 1 was C; wherein the X at position 2 was E;and wherein the X at position 7 was absent)) were reactive with serafrom a symptomatic, Aph-infected dog. At polypeptide concentrationsranging from 0.125 to 0.5 ug/ml, the strongest signal was obtained withSEQ ID NO:10 (p37-2).

Example 7 Detection of Aph Antibody by Polypeptide p37-2

An experiment was conducted to determine whether a synthetic polypeptidecomprising SEQ ID NO:10 (p37-2) can detect Aph antibodies in sera fromdogs exhibiting clinical symptoms of acute Anaplasmosis.

An ELISA was performed as described herein in Example 6, except thesynthetic peptide was coated at 0.5 ug/ml. The test samples were serafrom Aph infected dogs exhibiting clinical symptoms of acuteanaplasmosis. Four random samples from normal dogs were tested asnegative controls to determine a cut off OD value (cut off=mean+2×SD).Test samples producing OD values above this cutoff value were deemed“positives” and are represented by “+” in Table 6.

TABLE 6 ID P37-2 ME-485 + ME-487 + ME-560 + ME-562 + ME-568 + ME-601 +ME-616 + ME-631 + ME-661 + ME-670 + ME-699 + ME-703 + ME-710 + ME-716 +ME-734 + ME-741 + ME-748 − ME-753 + ME-476 + ME-534 + ME-630 + ME-639 −ME-653 − ME-691 − ME-700 + ME-746 +

Of the 26 samples tested, 22 samples were positive in the ELISA usingSEQ ID NO:10. These results demonstrate that a polypeptide comprisingSEQ ID NO:10 can detect Aph antibodies in sera from dogs having symptomsof acute anaplasmosis with high sensitivity.

Example 8 Early Detection of Aph Antibody in Sera of ExperimentallyInfected Dogs by Peptide p37-2

An indirect ELISA was conducted to determine the time course ofseroreactivity to peptide p37-2 (SEQ ID NO:10) following experimentalAph infection (Table 7). The assay was performed according to theprocedure as described herein in Example 6, except the synthetic peptidewas coated at 0.5 ug/ml. Canine sera were obtained from twoexperimentally infected dogs. One of these dogs was infected with Aphstrain Webster (a human isolate), and developed thrombocytopenia whichreached its worst levels at day 10. Lymph node swelling was firstdetected at day 7. The second dog was infected with Aph strain MN98E.4(a canine isolate), and developed thrombocytopenia which reached itsworst levels at day 14. Mild lymph node swelling was first detected atday 4, and fever peaked at day 9 in the second dog. Blood samples weretaken at various time points post-infection. Aph seroreactivity wasfirst detected at day 7 in sera from the dog infected with strainWebster, and at day 14 in sera from the dog infected with strain MN98E.4(Table 7). Thus, p37-2 can detect of Aph infection in dogs at about 7-14days post-infection. Since the onset of clinical symptoms of acuteanaplasmosis typically occurs within 1-2 weeks from the time ofinfection, p37-2 is useful as a diagnostic tool as early as the time ofonset of symptoms.

TABLE 7 days post- p37-2 ELISA Aph strain infection cut-off = 0.21Webster 0 − 2 − 4 − 7 + 10 + 14 + 17 + 21 + 24 + MN98E.4 0 − 2 − 4 − 7 −10 − 14 + 17 + 21 + 24 +

Example 9 Detection of Apl Antibody by Polypeptide p37-2

An experiment was conducted to determine whether a synthetic polypeptidecomprising SEQ ID NO:10 (p37-2) can detect Apl antibodies in sera fromtwo experimentally infected dogs. The assay was performed as describedherein in Example 6, except the synthetic peptide was coated at 0.5ug/ml. Samples from 10 normal dogs were tested as negative controls todetermine a cut off OD value (cut off=mean+2×SD). Test samples producingOD values above this cutoff value were deemed “positives” and arerepresented by “+” in Table 7.

TABLE 7 Days Canine Post ID Infection p37-2 A5 3 − 7 − 10 + 14 + 17 +21 + 24 + 28 + 35 + 42 + 49 + 56 + 63 + 71 + 79 + A6 3 − 7 − 10 + 13 +17 + 21 + 24 + 28 + 34 + 42 + 49 + 56 + 62 + 70 + 78 +

Sera from both dogs were positive starting at day 10 post-infection inthe ELISA using SEQ ID NO:10. These results demonstrate that apolypeptide comprising SEQ ID NO:10 can detect Apl antibodies in serafrom Apl infected dogs, as early as 10 days post-infection.

While the working examples provided herein were performed on sera fromdogs, a skilled artisan will appreciate that the compositions andmethods disclosed herein are readily applicable to the detection of Aphand/or Apl in other host species of Aph and/or Apl, such as for examplehumans, horses, cats, deer and ruminants.

We claim:
 1. A purified polypeptide that is at least 95% identical toSEQ ID NO:1, 9, or 11, wherein the purified polypeptide consists of lessthan about 150 contiguous naturally occurring Anaplasma phagocytophilumamino acids, wherein, optionally, the purified polypeptide is linked toan indicator reagent, an amino acid spacer, an amino acid linker, asignal sequence, a stop transfer sequence, a transmembrane domain, aprotein purification ligand, a heterologous polypeptide, one or moreadditional polypeptides comprising SEQ ID NO:1, 2, 6, 8, 9, 10, 11, 12,or a combination thereof.
 2. The purified polypeptide of claim 1,wherein the polypeptide comprises SEQ ID NO:1, 9, or
 11. 3. The purifiedpolypeptide of claim 1, wherein the purified polypeptide consists ofless than about 100 contiguous naturally occurring Anaplasmaphagocytophilum amino acids or wherein the purified polypeptide consistsof less than about 50 contiguous naturally occurring Anaplasmaphagocytophilum amino acids.
 4. A method of detecting antibodies thatspecifically bind an Anaplasma phagocytophilum polypeptide in a testsample, comprising: (a) contacting a purified polypeptide at least 95%identical to SEQ ID NO:1, 9 or 11, wherein the purified polypeptideconsists of less than about 150 contiguous naturally occurring Anaplasmaphagocytophilum amino acids, with the test sample, under conditions thatallow polypeptide/antibody complexes to form, wherein, optionally, thepurified polypeptide is linked to an indicator reagent, an amino acidspacer, an amino acid linker, a signal sequence, a stop transfersequence, a transmembrane domain, a protein purification ligand, aheterologous polypeptide, one or more additional polypeptides comprisingSEQ ID NO:1, 9, 11, or a combination thereof; (b) detecting thepolypeptide/antibody complexes; wherein the detection of thepolypeptide/antibody complexes is an indication that antibodies specificfor Anaplasma phagocytophilum are present in the test sample.
 5. Themethod of claim 4, further comprising contacting the complexes of step(a) with an indicator reagent prior to the performance of step (b). 6.The method of claim 4, wherein the amount of antibodies in the testsample is determined.
 7. The method of claim 4, wherein the purifiedpolypeptide is attached to a substrate.
 8. A method of detecting anAnaplasma phagocytophilum infection in a subject comprising: (a)contacting a purified polypeptide that is at least 95% identical to SEQID NO:1, 9 or 11, wherein the purified polypeptide consists of less thanabout 150 contiguous naturally occurring Anaplasma phagocytophilum aminoacids, with a biological sample from the subject under conditions thatallow polypeptide/antibody complexes to form, wherein, optionally, thepurified polypeptide is linked to an indicator reagent, an amino acidspacer, an amino acid linker, a signal sequence, a stop transfersequence, a transmembrane domain, a protein purification ligand, aheterologous polypeptide, one or more additional polypeptides comprisingSEQ ID NO:1, 9, 11, or a combination thereof; (b) detecting thepolypeptide/antibody complexes; wherein the detection of thepolypeptide/antibody complexes is an indication that the subject has anAnaplasma phagocytophilum infection.
 9. A composition comprising thepurified polypeptide of claim 1 and a pharmaceutically-acceptable orveterinarily acceptable carrier.
 10. The composition of claim 9, furthercomprising an adjuvant.
 11. An immunogen comprising a purifiedpolypeptide having at least 95% identity to SEQ ID NO:1, 9, or 11, andone or more additional regions or moieties covalently joined to thepolypeptide at the carboxyl terminus or amino terminus, wherein eachregion or moiety has at least one of the following properties: enhancesthe immune response, facilitates purification, or facilitatespolypeptide stability.
 12. The purified polypeptide of claim 1, whereinthe polypeptide is attached to a substrate.
 13. The purified polypeptideof claim 1, wherein the purified polypeptide consists of between 40 and100 naturally occurring Anaplasma phagocytophilum amino acids.
 14. Thepurified polypeptide of claim 1, wherein the purified polypeptideconsists of SEQ ID NO:1, 9, or 11, wherein, optionally, the purifiedpolypeptide is linked to an indicator reagent, an amino acid spacer, anamino acid linker, a signal sequence, a stop transfer sequence, atransmembrane domain, a protein purification ligand, a heterologouspolypeptide, one or more additional polypeptides comprising SEQ ID NO:1,2, 6, 8, 9, 10, 11, 12, or a combination thereof.
 15. The method ofclaim 4, wherein the purified polypeptide consists of SEQ ID NO:1, 9, or11, wherein, optionally, the purified polypeptide is linked to anindicator reagent, an amino acid spacer, an amino acid linker, a signalsequence, a stop transfer sequence, a transmembrane domain, a proteinpurification ligand, a heterologous polypeptide, one or more additionalpolypeptides comprising SEQ ID NO:1, 2, 6, 8, 9, 10, 11, 12, or acombination thereof.
 16. The method of claim 8, the purified polypeptideconsists of SEQ ID NO:1, 9, or 11, wherein, optionally, the purifiedpolypeptide is linked to an indicator reagent, an amino acid spacer, anamino acid linker, a signal sequence, a stop transfer sequence, atransmembrane domain, a protein purification ligand, a heterologouspolypeptide, one or more additional polypeptides comprising SEQ ID NO:1,2, 6, 8, 9, 10, 11, 12, or a combination thereof.